Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-l-Arabinofuranosidases

Article abstract of DOI:10.1021/jacs.9b11351

Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicell

Full text of DOI:10.1021/jacs.9b11351

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Article
Rational Design of Mechanism-Based Inhibitors and Activity-Based
Probes for the Identification of Retaining #-L-Arabinofuranosidases
Nicholas McGregor, Marta Artola, Alba Nin-Hill, Daniel Linzel, Mireille Haon, Jos Reijngoud,
Arthur F. J. Ram, Marie-Noelle Rosso, Gijsbert A. van der Marel, Jeroen D. C. Codée, Gilles
P. van Wezel, J-G Berrin, Carme Rovira, Herman S. Overkleeft, and Gideon J. Davies
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b11351 • Publication Date (Web): 13 Feb 2020
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Rational Design of Mechanism-Based Inhibitors and Activity-Based
Probes for the Identification of Retaining α-L-Arabinofuranosidases
Nicholas G.S. McGregor,a,† Marta Artola,b,† Alba Nin-Hill,c,† Daniël Linzel,b Mireille Haon,d Jos Reijngoud,e Arthur Ram,e Marie-
Noëlle Rosso,d Gijsbert A. van der Marel,b Jeroen D.C. Codée,b Gilles P. van Wezel,e Jean-Guy Berrin,d Carme Rovira,*,c,f Herman
S. Overkleeft,*,b Gideon J. Davies*,a
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aYork Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, York, YO10 5DD
bLeiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA Leiden, The Netherlands, cDepartament de
Química Inorgànica i Orgànica (Secció de Química Orgànica) & Institut de Química Teòrica i Computacional (IQTCUB),
Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain, dINRA, Aix Marseille University, Biodiversité et Bio-
technologie Fongiques (BBF), UMR1163, F-13009 Marseille, France. eMolecular Microbiology and Biotechnology, Institute
of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands, fInstitució Catalana de Recerca i
Estudis Avançats (ICREA), 08020 Barcelona, Spain.
†These authors contributed equally to this work.
* Corresponding authors
KEYWORDS: Activity-based protein profiling, cyclophellitol, secretome, glycoside hydrolase, -L-arabinofuranose
ABSTRACT: Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic
system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex
hemicellulosic and pectinaceous polysaccharides requires the production of α-L-arabinofuranosidases among a wide variety of co-
expressed carbohydrate-active enzymes. To selectively detect and identify α-L-arabinofuranosidases produced by fungi grown on
complex biomass, potential covalent inhibitors and probes which mimic α-L-arabinofuranosides were sought. The conformational
free energy landscapes of free α-L-arabinofuranose and several rationally designed covalent α-L-arabinofuranosidase inhibitors were
analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a
combination of kinetic measurements, intact mass spectrometry, and structural experiments, the designed inhibitors were shown to
efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-L-arabinofuranosidases. Activity-based probes elaborated
from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-L-arabinofuranosidases within
the secretome of A. niger grown on arabinan. This method was extended to the detection and identification of α-L-arabino-
furanosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the
cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of
complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-L-arabinofuranosidases.
Inspired by the work of Withers3–5 and Wright6, we have been de-
veloping cyclophellitol-derived activity-based inhibitors and
INTRODUCTION
probes (some aspects of which are reviewed in 7–9) for the rapid
Carbohydrate-degrading machinery is a fundamentally important
detection and identification of specific biomass-degrading glyco-
component of the metabolic systems that underpin the global car-
side hydrolases within complex systems. The potential of cy-
bon cycle. Our understanding of these systems is dependent on an
clophellitol-derived activity-based probes (ABPs) as tools for the
ability to identify the capacities of the carbohydrate-active enzymes
detection and identification of retaining glycoside hydrolases has
produced by an organism. The growth of genomic libraries has re-
been well-established.10 Mimicking the half chair conformation of
vealed an expansive world of carbohydrate-degrading enzymes, of
the enzymatic transition state, cyclophellitol and cyclophellitol
which only a small fraction have been isolated and probed for cat-
aziridine derivatives react specifically with the catalytic nucleo-
alytic potential.1 Transcriptomic and proteomic experiments com-
phile of a retaining glycoside hydrolase, forming a non-hydrolyza-
paring the gene expression and protein secretion patterns of organ-
ble ester linkage through a ring-opening addition.11 This general
isms grown on different substrates have helped to identify the ge-
strategy has been exploited to inhibit and label glycosidases dis-
netic logic used by these organisms to efficiently degrade recalci-
playing a variety of specificities including α- and β-D-gluco-
trant biomass.2 However, the underlying chemical rationale for
sidases12–14
,
β-D-glucuronidases15, and α- and β-D-galacto-
these expression patterns remains obscure without highly detailed
experimental work characterizing the role of each enzyme.
sidases16,17 among others. Building on this work, we have recently
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reported the synthesis and validation of a collection of cyclophel-
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Secretome production
litol-derived inhibitors and probes which specifically label retain-
ing β-D-xylanases and β-D-xylosidases.18 These compounds were
able to efficiently attach chemical handles for the detection and
identification of key secreted xylan-degrading enzymes within an
Aspergillus secretome. Expanding this toolbox to target side-chain
removal enzymes has remained a challenge, not least for
furanoside-active enzymes.
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Aspergillus niger strain N402 was grown as described by
Schröder et al.18 with a mixture of 50 mM arabinose, 1% sugar
beet arabinan, and 2 mM fructose as the sole carbon source.
Samples were collected, 0.2 µm-filtered, and snap-frozen after
5 days. Samples were stored at -80°C until being thawed imme-
diately before use.
α-L-Arabinofuranoside “side-chains” are commonly found on both
hemicellulosic and pectinaceous plant polysaccharides. The effi-
cient removal of α-L-arabinofuranose branches enhances the break-
down of xylan-rich biomass.19 Furthermore, α-L-arabino-
furanosidases are an essential part of the polysaccharide utilization
loci which ferment arabinan chains in dietary rhamnogalacturonan
I and arabinogalactan within the human gut.20 Thus, cyclophellitol-
derived ABPs and inhibitors for α-L-arabinofuranosidases could be
used to identify the enzymes responsible for the breakdown of a
variety of complex polysaccharides. However, it is not currently
known whether cyclophellitol derivatives can be effectively ex-
tended to target furanosidases.
The strains Abortiporus biennis BRFM 1215 (A. biennis),
Fomes fomentarius BRFM 1323 (F. fomentarius), Hexagonia
nitida BRFM 1328 (H. nitida), Leiotrametes menziesii BRFM
1557 (L. menziesii), Polyporus brumalis BRFM 958 (P.
brumalis), Trametes ljubarskyi BRFM 957 (T. ljubarskyi)
Trametes gibbosa BRFM 952 (T. gibbosa), and Trametes mey-
enii BRFM 1361 (T. meyenii) were obtained from the CIRM-CF
collection (International Centre of Microbial Resources dedicated
to Filamentous Fungi, INRA, Marseille, France). All strains were
identified by morphological and molecular analysis of ITS (Internal
Transcribed Spacer) sequences. The strains were maintained on
malt agar slants at 4°C.
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No route to the synthesis of covalent inhibitors of α-L-arabino-
furanosidases has previously been identified. The first synthesis of
covalent furanose-configured inhibitors was the preparation of β-
D-arabinofuranosyl and α-L-xylofuranosyl aziridines reported by
Bols et al. in 2003.21 These were prepared via N-O reduction of
cyclopentaisoxazolidines. Due to the inverted stereochemistry of
the electrophilic moiety with respect to C4 (carbohydrate number-
ing), this synthetic strategy cannot be translated to α-L-arabino-
furanose analogues, so new synthetic methodologies are needed to
expand the scope of synthetically accessible furanoside mimics.
Basidiomycete cultures were grown in 250 mL baffled Erlenmeyer
flasks with 100 mL medium containing 2.5 g L−1 of maltose as a
starter (except for the maltose control condition; 20 g L−1), 1.842 g
L−1 of diammonium tartrate as a nitrogen source, 0.5 g L−1 yeast
extract, 0.2 g L−1 KH2PO4, 0.0132 g L−1 CaCl2/2H2O and 0.5 g L−1
MgSO4/7H2O, and as a main carbon source, 15 g L−1 (dry weight)
of wheat straw (Triticum aestivum) or Wiley-milled aspen (Populus
grandidentata). Cultures were incubated in the dark at 30°C with
shaking at 120 rpm. The cultures were stopped 10 days after inoc-
ulation and the culture broths (secretomes) were filtered using 0.2
μm polyethersulfone membrane (Millipore) and then stored at -
20°C until use.
We have designed a collection of putative α-L-arabinofuranosidase
inhibitors and ABPs with different electrophilic traps and detection
tags. Potential inhibitors were analyzed in silico for their ability to
mimic the natural 5-membered ring structure, stereochemistry, and
conformational itinerary of retaining α-L-arabinofuranosides.
These inhibitors and probes were synthesized following a general
route inspired by the synthesis of six-membered cyclophellitol de-
rivatives. Inhibition kinetics measured with α-L-arabino-
furanosidases from glycoside hydrolase families 51 and 54 (GH51
and GH54), the two major families of retaining α-L-arabino-
furanosidases, were measured to validate our predictions. Further-
more, the ability of our α-L-arabinofuranosidase probes to facilitate
the selective detection, identification, and characterization of active
GH51 and GH54 enzymes within the complex mixture of enzymes
secreted by Aspergillus niger was validated. These methods were
then extended to the identification of α-L-arabinofuranosidases
within the secretomes of basidiomycete fungi grown on complex
biomass.
Recombinant enzyme production
The coding sequence for Geobacillus stearothermophilus abfA
(GsGH51, GenBank: AAD45520) was synthesized with E. coli
codon optimization and cloned into pET28a(+) with an N-ter-
minal TEV protease-cleavable 6xhis tag by GenScript. Follow-
ing transformation of BL21(DE3) Gold, the enzyme was pro-
duced in an auto induction medium (1% tryptone, 0.5% yeast
extract, 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM NH4Cl, 5
mM Na2SO4, 0.05% glucose, 0.5% glycerol, 0.2% lactose) at
37°C. The enzyme was purified as described previously22 with
an added overnight treatment with his-tagged TEV protease
S219V23 in pH 8 Tris-HCl, 5 mM DTT, 1 mM EDTA at RT
followed by inverse histrap purification and desalting into 5
mM Tris-HCl, 1 mM EDTA, pH 8.0.
Aspergillus niger abfA (AnAbfA, GenBank: CAK43424) and
Aspergillus kawachii abfB (AkAbfB, GenBank: BAB96816)
were produced in P. pastoris X-33. A plasmid encoding
AkAbfB in pPICZα with no purification tag was obtained from
professors Takuya Koseki and Shinya Fushinobu. AnAbfA was
synthesized by IDT as a GBlock and cloned into the vector frag-
ment PCR-amplified from the AkAbfB-pPICZa plasmid using
Gibson assembly24. The AkAbfB (E221Q) mutant was gener-
ated using the Q5 site-directed mutagenesis kit (New England
Biolabs) with primers designed by the NEBaseChanger tool.
EXPERIMENTAL
All chemicals were purchased from Sigma-Aldrich unless oth-
erwise specified.
Design and synthesis of α-L-arabinofuranose-configured
cyclophellitol derivatives
Detailed protocols for synthesis of compounds 1 to 23 and their
NMR characterization can be found in the supplementary ma-
terials.
Plasmid DNA for transformation into P. pastoris was linearized
with SacI and purified using a PCR clean-up kit (Qiagen) using
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heated to 95°C for 5 minutes and 10 μL was separated
ultrapure water as the eluent. 100 ng of linearized DNA was
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electroporated into 80 µL of X-33 electrocompetent cells pre-
pared following the protocol of Wu and Letchworth.25 Nine col-
onies from each transformation were purified on YPD-Zeocin
plates, then grown in 5 mL of BMGY medium. At saturation
(OD600 ~ 20) cells were collected by centrifugation and re-sus-
pended in 5 mL of BMMY medium for expression screening at
20°C. The transformant which gave the highest titer of the tar-
get protein with minimal detectable contamination after 3 daily
0.5% MeOH feedings was grown in 500 mL of BMGY in a 2.5
L baffled shaking flask at 30°C overnight. The culture was then
cooled to 20°C and supplemented with 2.5 mL of 100% MeOH
each day for 3 days.
through a 4-15% Criterion (Bio-Rad) gel.
For scaled up labelling, 20 µL of 60 µM ABP 4 was added to
100 µL of buffered secretome and incubated at 30°C for 1
hour. 500 µL of acetone was then added and the samples
were incubated at -20°C for 1 hour. Precipitate was col-
lected by centrifugation at 10000 x g for 5 minutes at 4°C.
The supernatant was discarded, and the sample was left to
air dry to minimize residual acetone. The sample was then
re-suspended in 20 µL of 1X SDS-PAGE loading dye and
heated to 95°C for 5 minutes to dissolve. The entire sample
was then separated through a 4-20% gel.
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The culture supernatant was clarified by centrifugation fol-
lowed by 0.45 μm-filtration. 500 mL of medium was concen-
trated using a KrosFlo tangential flow system fitted with a 30
kDa MWCO mPES filter, then diluted with 9 volumes of 10
mM pH 5 sodium acetate buffer and concentrated again. Protein
was then collected onto a 5 mL Q sepharose HP column (GE
Healthcare), washed with 3 CV of 50 mM pH 5 sodium acetate
buffer, then eluted with a 25 CV gradient from 0 to 0.5 M NaCl
in the same buffer. Fractions from the largest UV-active peak
were pooled, concentrated to 10-30 mg/mL using a 30 kDa
MWCO centrifugal concentrator (Amicon) and purified over
Superdex 200 (GE Healthcare) into 50 mM sodium acetate pH
5. Protein-containing fractions were pooled and concentrated to
give a colorless 15-25 mg/mL protein solution. ~5 mg of protein
was then treated with 1000 U of EndoHf (New England Bi-
olabs) overnight at RT. This was purified using a 5 mL Q se-
pharose HP column as above. To prepare the sample for crys-
tallization, the eluent from Q sepharose was mixed 1:1 with sat-
urated ammonium sulfate and purified over a 1 mL phenyl se-
pharose HP column with a 25 CV gradient from 2 M ammonium
sulfate to 0 M ammonium sulfate in 50 mM pH 5 sodium acetate
buffer. Protein-containing fractions were pooled, desalted into
20 mM sodium acetate pH 5, concentrated to 10-30 mg/mL and
frozen at -80°C.
In situ characterization of secreted enzymes
The pH optimum of enzyme labelling was determined by visu-
alization with ABP 4 using the standard protocol (above) with
variable buffer solutions including a series of McIlvane buffers
prepared at 0.5 M strength (0.28 M citrate, 0.22 M phosphate)
from pH 2-7.5 in 0.5 pH unit increments and a series of succin-
ate-phosphate-glycine (SPG) buffers prepared at 0.5 M strength
(62.5 mM succinic acid, 219 mM phosphate, 219 mM glycine)
from pH 4-10 in 1 pH unit increments. 5 µL of each buffer was
added to 45 µL of A. niger arabinan secretome immediately
prior to ABP addition.
The thermal tolerance of secreted enzymes was assayed at the
inhibition optimum (50 mM pH 6.5 phosphate buffer) by incu-
bating the A. niger arabinan secretome at temperatures ranging
from RT to 95°C for 1 hour. Secretome samples were then rap-
idly cooled to 20°C and enzymes were visualized with ABP 4
using the standard protocol.
Measuring irreversible inhibition kinetics
The kinetics of enzyme inhibition were measured using a con-
tinuous assay26,27 at 25°C in a 384-well plate with 4-
methylumbelliferyl α-L-arabinofuranoside (4MU-Araf) as sub-
strate. Kinetic measurements were made in technical quadrupli-
cate. Curve fitting and statistical analysis was performed using
OriginPro graphing software. Enzymes were diluted in 50 mM
sodium phosphate buffer pH 7.0. Substrate was dissolved in
DMSO to give a 100 mM stock which was diluted with ul-
trapure water. Putative inhibitors were dissolved in and diluted
with ultrapure water with the exception of inhibitor 3 which was
dissolved in DMSO to give a 50 mM stock, which was diluted
with ultrapure water.
Enzyme visualization with ABP 4
ABP 4 was dissolved in DMSO to prepare a 10 mM stock solu-
tion which was diluted in ultrapure water. Unless otherwise
noted, samples were stained with 10 μM ABP 4 at 37°C for 30
minutes at pH 6.5 and proteins were separated at 200 V using
either a precast 4-20% (Bio-Rad) or an 8.75% 1 mm minipro-
tean SDS-PAGE gel. Fluorescence was imaged using a Ty-
phoon 5 laser scanner with the Cy5 laser and filter set. Enzyme
molecular weights were estimated using a Pageruler 10-180
kDa pre-stained protein ladder.
Enzyme specific activity was initially assessed by monitoring
the hydrolysis of 50 μM 4MU-Araf in pH 7 phosphate for 10
minutes. Michaelis-Menten parameters for the hydrolysis of
4MU-Araf were estimated by varying the substrate concentra-
tion from 4 to 500 μM and fitting a site-saturation kinetic model
(v0/[E]t = kcat[S]0/KM+[S]0) to the resulting rate vs. substrate
concentration data (Supplemental Table 1, Supplemental Figure
1A, Supplemental Figure 2A). Measurements were made at an
excitation wavelength of 390 nm (15 nm bandwidth) to elimi-
nate primary inner filter effects at substrate concentrations as
high as 500 μM in our assay format (Supplemental Figure 3).
Inhibition kinetics were measured using a substrate concentra-
Basidiomycete secretomes were buffered with 0.1 volumes of 1
M NH4OAc pH 5.5. For screening, 17.2 µL of buffered secre-
tome was mixed with 2.8 µL of 60 µM ABP 4 and incubated
for 1 hour at 30°C. The sample was then supplemented with
2 μL of 10X glycoprotein denaturing buffer (New England
Biolabs), heated to 95°C for 5 minutes and split in half.
Each half was mixed with 10 µL of 2x PNGaseF mastermix
(2X glycobuffer 2, 2% NP-40 containing either 0 or 7.5 U/µL
of PNGaseF) and incubated for 1 hour at 37°C. Samples
were then diluted with 6.7 µL of 4X SDS-PAGE loading dye,
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tion of 100 μM and an excitation wavelength of 360 nm, an en- (Supplemental Figure 5A). To generate inhibitor-bound com-
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4
5
6
7
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zyme concentration of 50 ng/mL, and variable inhibitor concen-
trations. Each fluorescence vs. time curve was fitted with an ex-
plexes, crystal-containing droplets were supplemented with 0.1
µL of 2 mM inhibitor in water and incubated overnight prior to
cryo-protection in well solution supplemented with 12.5% glyc-
erol and flash freezing in LN2.
k
t
ponential decay model (F = F∞(1-e- )). The resulting apparent
decay constants were plotted against inhibitor concentration
and fitted with a site-saturation kinetic model with correction
for competition by the substrate using the measured KM value
app
Initially, crystals of AkAbfB were grown essentially as de-
scribed by Miyanaga et al.28 Optimized crystals grew from 0.5
μL of 10 mg/mL AkAbfB in 50 mM pH 5 sodium acetate mixed
with 0.5 μL of 100 mM Tris-HCl pH 8.0, 200 mM MgCl2, 400
mM NaCl, 20% PEG6000, 2.5% DMF at 279 K. However, pref-
erential formation of poor-quality needle clusters and poor dif-
fraction of these crystals led us to explore other crystallization
conditions. EndoH-deglycosylated AkAbfB or AkAbfB
(E221Q) (12 mg/mL in 50 mM sodium acetate pH 5.0) formed
slow-growing isolated crystals when mixed 2:1 with 0.2 M lith-
ium sulfate, 0.1 M sodium acetate pH 4.5, 50% PEG400
(Supplemental Figure 5B). Supplementation with 0.2-0.5 M
NaCl resulted in more rapid crystal growth. To generate inhibi-
tor-bound complexes, crystals were transferred to mother liquor
supplemented with inhibitor 6 or 2 to a final concentration of
0.2 mM, or saturated with PNP-Araf (for AkAbfB (E221Q)).
Crystals were soaked for 1 hour at RT prior to freezing.
and the initial substrate concentration (kapp
=
kin-
act[I]0/1+([S]0/KM)+([I]0/KI)).
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Intact MS following enzyme labelling
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GsGH51 or EndoH-treated AkAbfB were diluted to 0.1 mg/mL
in their respective SEC elution buffers. Compounds 1, 2, or 6
were added to a final concentration of 50 μM and incubated for
30 minutes at RT. The treated protein samples were diluted with
4 volumes of 1% formic acid, 10% acetonitrile and 5 µL was
injected over an MSPac DS-10 Desalting Cartridge flowing at
30 μL/min using a NanoAcquity HPLC (Waters). Following a
5-minute wash with 20% acetonitrile, 0.1% formic acid in wa-
ter, protein was eluted into a maXis UHR-Tof (Bruker) with a
10-minute gradient from 20-55% acetonitrile. The column was
washed for 2 minutes with 80% acetonitrile and equilibrated for
3 minutes with 20% acetonitrile between runs. Following pro-
tein signal integration and baseline subtraction, spectra were de-
convoluted using the maximum entropy algorithm within
COMPASS to calculate protein mass.
Diffraction data were collected at Diamond Light Source (Har-
well, UK) on beamline I04 and automatically processed using
the fast_dp29 (GsGH51), autoPROC30 (AkAbfB-2 and AkAbfB-
6), or Xia231 (AkAbfB-PNP-Araf) pipelines. Computation was
carried out using programs from the CCP4 suite32 unless other-
wise stated. All crystal structure figures were generated using
Pymol (Schrodinger). Data collection and processing statistics
for all structures are given in Supplemental Table 2.
Enzyme pull-down using ABP 5
A. niger arabinan secretome was buffered with 50 mM McIl-
vane buffer pH 6.5, then treated with 0.1 mM inhibitor 2 or
DMSO control for 1 hour at 37°C (inhibitor 6 is also suitable
for pre-treatment, Supplemental Figure 4). Following this, the
secretome was treated with either 20 µM ABP 5 or DMSO con-
trol for 30 minutes at 37°C. Biotinylated proteins were pulled
down, digested, and identified as described by Schröder et al.18
Basidiomycete secretome samples were processed without con-
centration or lyophilization with three modifications to the pro-
tocol: firstly, protein was precipitated through the addition of 4
volumes of acetone followed by incubation at -20°C for 1 hour;
secondly, following the initial strep mag sepharose bead wash
with 0.5% SDS, beads were washed with 2% SDS at 65°C for
10 minute with agitation followed by 2 M urea and then PBS;
and lastly, peptides liberated through on-bead digest were mod-
ified with TMT0 following the manufacturer’s instructions
prior to LC-MS/MS analysis using an Orbitrap Fusion Tribrid
mass spectrometer (Thermo Scientific). Peptides were identified
by mapping onto the predicted proteomes deduced from genome
sequence of A. biennis BRFM 1778, F. fomentarius BRFM
1823, L. menziesii BRFM 1781, and T. gibbosa BRFM 1770.
For each genome (to be published elsewhere), CAZymes were an-
notated as in Lombard et al., 20141. All genome and proteome data
are publicly available on the Mycocosm portal (www.my-
cocosm.jgi.doe.gov).
Structure solution and refinement
Data for GsGH51 bound to inhibitors 2 and 6 were collected to
1.40 Å. Each structure was solved by molecular replacement
using Phaser33 with the known structure (PDBID: 1pz3) as the
search model. The resulting solution showed clear density for
the bound ligand within the enzyme active site. Ligand coordi-
nates and dictionaries were generated using jLigand34 and built
into the model using Coot35, followed by alternating rounds of
manual model building and refinement using Coot and
REFMAC536.
Data for AkAbfB bound to inhibitors 2 and 6 were collected to
1.47 and 1.86 Å, respectively. Each structure was solved by mo-
lecular replacement using Phaser with the known structure
(PDBID: 1wd3) as the search model. The resulting solution
showed clear density for the bound ligand within the enzyme
active site. The structures were refined, as above, and the same
ligand coordinates and geometries were used.
Data for AkAbfB (E221Q) bound to PNP-Araf were collected
to 1.64 Å. The structure was solved by molecular replacement
using Phaser with the AkAbfB-2 complex as the search model.
The resulting structure showed clear density for two PNP-Araf
(ligand ID: KHP) molecules bound to the carbohydrate-binding
module. Following several rounds of manual model building
and refinement, partial density for an additional PNP-Araf mol-
ecule, which was modelled at 60% occupancy, became apparent
in the active site.
Enzyme crystallization and diffraction
Crystals of GsGH51 were grown essentially as described by
Hövel et al.22 Optimized crystals were grown by mixing 1.2 µL
of protein (10 mg/mL in 5 mM Tris-HCl pH 8.0) with 0.6 µL of
well solution containing 15% PEG3350, 5% 2-propanol, 0.1 M
Tris-HCl pH 7.5, 0.80 M NH4F in a sitting drop at 293 K
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with an integration time step of 2 fs, was used. The binding free
energy of the compounds were obtained by using the MMPBSA
Conformational analysis
1
2
3
4
5
6
7
8
Conformational free energy landscapes (FELs) were computed
for α-L-arabinofuranose and compounds 1, 2, and 6 using Den-
sity Functional Theory-based molecular dynamics (MD), ac-
cording to the Car-Parrinello (CP) method.37 Each molecule
was enclosed in an isolated cubic box of 12.5 Å × 12.5 Å × 12.5
Å. A fictitious electron mass of 500 atomic units (a.u.) was used
for the CP Lagrangian and a time step of 0.12 fs was used in all
CPMD simulations to ensure that the adiabacity of the fictitious
kinetic energy of the electrons was smaller than 10-5 a.u./atom.
The Kohn-Sham orbitals were expanded in a plane wave basis
set with a kinetic energy cut-off of 70 Ry. Ab initio pseudopo-
tentials, generated within the Troullier-Martins scheme, were
employed.38 The Perdew, Burke, and Ernzerhoff generalized
gradient-corrected approximation39 was selected in view of its
good performance40 in previous work on isolated sugars41, gly-
cosidases, and glycosyltransferases42. The metadynamics algo-
rithm43, provided by the Plumed 2 plugin44, was used to explore
the conformational free energy landscape of the systems, taking
as collective variables the pseudorotational phase (φ) puckering
coordinate45,46, as well as a dihedral angle accounting for the
rotation of the sugar hydroxymethyl group. The energy was pro-
jected into the φ coordinate for representation purposes. Ini-
tially, the height of these Gaussian terms was set at 0.6 kcal/mol
and a new Gaussian-like potential was added every 500 MD
steps. Once the whole free energy space was explored, the
height of the Gaussian terms was reduced to 0.2 kcal/mol to fa-
cilitate convergence of the FEL. The width of the collective var-
iables was set according to their oscillations in the free dynam-
ics which corresponded to 0.035 and 0.1 rad for φ and the hy-
droxymethyl dihedral angle, respectively. The simulations were
stopped when energy differences among wells remain constant,
which was further confirmed by a time-independent free energy
estimator.47 The exploration of the phase space was extended up
to 380, 360, 324, and 474 ps for α-L-arabinofuranose, com-
pound 1, compound 2, and compound 6, respectively. The er-
rors in the principal minima, taken as a standard deviation (SD)
from the last 200 ps, are below 0.6 kcal mol-1. Conformational
FELs computed using only φ as CV gave very similar results.
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The Michaelis complexes of compounds 1, 2, and 6 were mod-
elled using the crystal structures of the adducts obtained for
GsGH51 and AkAbfB as a reference. In the case of compounds
1 and 2, the Michaelis complex was reconstructed by removing
the covalent bond between the inhibitor and the nucleophile in
the protein structure bound to inhibitor 2. The amine group was
reverted to an aziridine (compound 2), which was replaced with
an oxygen atom to give compound 1.
Molecular dynamics (MD) simulations were set up employing
the program LEaP included in the Amber suite48 and the ff14SB
protein force field49. The compounds were parametrized using
gaff250. The systems were solvated with explicit TIP3P water
molecules.51 They were neutralized with 31 and 21 sodium at-
oms for all neutral compounds in GsGH51 and AkAbfB, re-
spectively. The systems with protonated compound 2 were neu-
tralized with one fewer sodium atom (30 and 20 in GsGH51 and
AkAbfB, respectively). MD simulations were performed using
Amber1648. A thermal equilibration to 300 K was done prior to
the equilibration of dynamics in the NPT ensemble with a pro-
duction phase of 51 ns for each system. The SHAKE algorithm,
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method52
integrated
in
the
Amber
suite.
This approach has recently been successful in predicting the
performance of pyranose-like inhibitors.14,18
1
2
3
4
5
6
7
8
9
In contrast to GHs which act on pyranosides (e.g. α/β-gluco-
sidases53 and α/β-mannosidases54), little is known about the cat-
alytic conformational itineraries of α-L-arabinofuranosidases.
The computed FEL of α-L-arabinofuranose (Figure 1B) shows
that all conformations lie in an energy window of ≈ 5 kcal/mol.
This window is significantly narrower than what is typical for
pyranose compounds (≈ 15 kcal/mol)41,55 and shows that most
α-L-arabinofuranose conformations are thermally accessible.
1
10
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The most stable conformation is T2. However, this confor-
mation is not catalytically competent since the axial 2-OH
group creates steric hindrance with the nucleophile residue lo-
cated on the “beta” face of the sugar. Conformations between
2E and 4E, being only ≈ 2 kcal/mol higher in energy, feature an
equatorial 2-OH, eliminating this steric hindrance. Thus, the
ideal Michaelis complex conformation for an α-L-arabino-
2
4
furanosidase should be between E and E (shaded region in
Figure 1B).
To determine where on this landscape the observed confor-
mations of enzyme-bound species lie, we surveyed all of the
conformations of L-arabinofuranose observed within the active
sites of crystallized GH51 and GH54 enzymes. Specific α-L-
arabinofuranosidases have been identified within GH families
43, 51, 54, and 62, of which only families 51 and 54 follow the
anomeric stereochemistry-retaining Koshland double-displace-
ment mechanism.
The most detailed studies of α-L-arabinofuranosidase mecha-
nisms have been performed using bacterial GH51 enzymes.
Paes et al. obtained the structure of an intact branched penta-
saccharide substrate bound to the active site of TxAbf, a ther-
mostable GH51 from Thermobacillus xylanilyticus (PDB ID
2VRQ).56 Hövel et al. reported the crystal structure of Geo-
bacillus stearothermophilus AbfA (hereafter referred to as
GsGH51) bound to 4-nitrophenyl α-L-arabinofuranoside (PNP-
Araf) (PDB ID 1QW9).57 In both of these Michaelis complexes,
4
the α-L-arabinofuranose rings were found in the E confor-
mation (Figure 2A). Therefore, similar to observations with
GHs acting on pyranose sugars53, furanosidases distort the -1
sugar to a conformation that is pre-activated for catalysis. Thus,
the conformational catalytic itinerary for the rate limiting step
of the reaction for GH51 family is expected to go through an
oxocarbenium ion-like E3 conformer to fulfill the requirement
of having C4-O5-C1-C2 planarity.58
Figure 1. A) Graphical representation of the conformations of a 5-membered
ring according to the Cremer-Pople angle ϕ. B) Conformational FEL of iso-
lated α-L-arabinofuranose. Conformations observed in Michaelis complexes
of α-L–arabinofuranosidases are represented with a red star (PDB 2VRQ and
1QW9 for GH51 and PDB 6SXR, this work, for GH54). The conformational re-
gion having an equatorial O2 is shaded. C) Conformational FEL of α-L-arabi-
nofuranose-configured cyclophellitol (1), aziridine (2) and cyclic sulfate (6).
Beyond the bacterial GH51 enzymes, there is only one retaining
α-L-arabinofuranosidases which has been crystallized. The
structure of Aspergillus kawachii AbfB (a member of GH54
hereafter referred to as AkAbfB) with arabinose in the active
site (PDB ID 1WD4), displays a product complex ring confor-
mation of 4E.59 Unfortunately, no Michaelis complex of this en-
zyme had been reported to date.
RESULTS AND DISCUSSION
The free energy landscape of α-L-arabinofuranose, and the
conformational itinerary of family GH51 and GH54 retain-
ing α-L-arabinofuranosidases
To gain insight into the ability of our potential inhibitors to
mimic the natural conformational preferences of α-L-arabino-
furanosides, we computed the relative energy of all ring confor-
mations of compounds 1, 2, and 6. α-L-arabinofuranose was
also analyzed for comparison. The conformational free energy
landscape (FEL) of each molecule was calculated using ab ini-
tio metadynamics and the Cremer-Pople puckering coordinates.
Determination of the Michaelis complex of AkAbfB
To complete, and thus compare the conformational itineraries
of the GH51 and GH54 families, we studied AkAbfB as a model
GH54 active site. To observe the Michaelis complex, we soaked
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2
crystals of deglycosylated AkAbfB E221Q in a saturated solu-
pass through an E3 transition state conformation to give a E
glycosyl-enzyme intermediate. Following exchange of the leav-
ing group with water, the glycone then passes through a second
E3 transition state to form a lower energy product-bound com-
plex observed in the low energy E3 – 4T3 region. Therefore, the
predicted conformational itinerary for the two half-reactions is
4E → [E3]‡ → 2E (glycosylaton) and 2E → [E3]‡ → E3/4T3 (de-
glycosylation), as shown in Figure 2.
1
2
3
4
5
6
7
8
tion of PNP-Araf in mother liquor. The resulting 1.64 Å crystal
structure contained 3 PNP-Araf molecules: two full occupancy
molecules bound to the carbohydrate binding module and a par-
tial occupancy molecule bound in the active site (Supplemental
Figure 6A).
Overall, the Michaelis complex displayed similarity to the prod-
uct complex published by Miyanaga et al. in 200459
(Supplemental Figure 6B). O2 formed hydrogen bonds with the
carbonyl oxygen of Q221 and the backbone amide of D297. O3
formed hydrogen bonds with the backbone amide of G296 and
the carboxylate of D219. The ring oxygen formed a hydrogen
bond with the backbone amide of N222 and O5 formed hydro-
gen bonds with the carboxylate of D219 and the backbone am-
ide of N223. The furanose ring was found in a 4E conformation,
stacked against a hydrophobic surface formed by W206 and the
C176-C177 disulfide linkage. The axial nitrophenyl leaving
group pointed out of the active site into a solvent channel. The
electrophilic carbon (C1) was positioned 3 Å away from the
amide nitrogen, primed for migration away from the nitro-
phenyl leaving group with support from anti protonation of the
glycosidic oxygen by D297, the general acid/base.
Conformational analysis of potential α-L-arabino-
9
furanosidase inhibitors
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Having ascertained the FEL for α-L-arabinofuranose and the
conformational itinerary of retaining α-L-arabinofuranosidases,
we next considered the design and synthesis of covalent inhibi-
tors. As discussed above, both GH51 and GH54 enzymes form
Michaelis complexes in the 4E conformation (red stars in Figure
1B). Therefore, a suitable covalent α-L-arabinofuranosidase in-
4
hibitor should readily adopt a E conformation in which the
atom that mimics the anomeric carbon is similarly accessible
for nucleophilic attack from the beta face of the sugar ring.
Computed FELs for compounds 1, 2, and 6 (Figure 1C) show
that these conformations are energetically favored for 6,
whereas 1 and 2 instead prefer conformations in which the 2-
OH is axial (in the 1T2 – E2 – 3T2 region). Thus, cyclic sulfate 6
was anticipated to be a potentially more potent inhibitor than
the epoxide (1) or aziridine (2) for both GH51 and GH54 α-L-
arabinofuranosidases.
Based on this result, and the general observation of one itinerary
per family (at least for members active on similar substrates),54
we infer that enzymes within GH54 and GH51 share a common
catalytic conformational itinerary (Figure 2A). Following bind-
4
ing in a reactive E conformation, the glycone is predicted to
Figure 2. A) Koshland double-displacement mechanism employed by retaining α-L-arabinofuranosidases, as proposed for GH51 and GH54, showing the
conformational reaction itinerary including the (left-to-right) Michaelis complex, transition state 1, covalent substrate-enzyme intermediate, transition state 2, and the
hydrolyzed product. B) Chemical structures of putative α-L-arabinofuranosidase inhibitors 1, 2, 3, and 6, and ABPs 4 and 5.
hexene starting materials. α-L-arabinofuranose-configured cy-
clopentene was prepared in nine steps from commercial methyl
α-D-galactopyranoside in 15% yield. The initial installation of
a p-methoxybenzylidene acetal (PMP) at C4 and C6 of methyl
α-D-galactopyranoside (carbohydrate numbering) by treatment
with anisaldehyde dimethylacetal followed by benzylation at
C2 and C3 to afford intermediate 7 gave 74% yield over 2 steps
Synthesis of α-L-arabinofuranose-configured inhibitors
and ABPs
To synthesize α-L-arabinofuranosidase inhibitors, we took in-
spiration from the synthesis of six-membered cyclophellitol de-
rivatives beginning from appropriately functionalized cyclo-
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(Scheme 1). Selective opening of the PMP-group in compound
Page 8 of 20
1
2
3
4
5
6
7
8
7 with Bu2BOTf and BH3∙THF, followed by nucleophilic sub-
stitution of the primary alcohol with iodine and Vasella frag-
mentation with activated zinc powder afforded intermediate 10
in 60% yield over three steps. We were able to scale this process
up to 56 mmol with moderate yields. Wittig olefination of alde-
hyde 10 and subsequent ring-closing metathesis (RCM) with
second generation Grubb’s catalyst afforded 11. The PMB
group was then selectively removed with DDQ and intermedi-
ate 14 was obtained in 66% yield over 4 steps by subsequent
alkylation with freshly synthesized Bu3SnMeI. The key step, a
Wittig-Still rearrangement of intermediate 14 with n-BuLi at -
78°C, afforded the desired cyclopentene 15 in 68% yield.
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60
Scheme 2: Synthesis of epoxides. Reagents and conditions: a) m-CPBA,
DCM, 50°C, 18 h, 62%, 3.4:1 of 16:17. b) m-CPBA, DCM, 0°C, 4 days, 91%,
4.3:1 of 16:17. c) m-CPBA, DCM, 50°C, 18 h, 62%, 1:2 of 19:20 d) H2,
Pd(OH)2, MeOH, 18 h, 50%.
Taking advantage of the C2 and C4 stereochemistry of 18, di-
rect aziridination aided by steric hindrance of the vicinal pro-
tecting groups was attempted first. No aziridination was ob-
served with 3-amino-2-(trifluoromethyl)quinazolin-4(3H)-one
(Q-CF3) as nitrogen donor and phenyliodine(III) diacetate
(PIDA) to form the reactive acetylated quinazolinone.60 O-(2,4-
dinitrophenyl)hydroxylamine (DPH) and a ruthenium catalyst
also gave no aziridination.61 Hypothesizing that the alkene is
not accessible enough due to the conformation of cyclopentene
and/or steric hindrance of the benzyl groups, we pursued aziri-
dine 2 by benzylation of the primary hydroxyl of epoxide 16
and subsequent SN1 ring opening with sodium azide. This af-
forded two separable regio-isomers in 1:2 (21:22) ratio with
77% yield (Scheme 3). Hydroxyls of 21 and 22 were first tosyl-
ated and subsequently treated with triphenylphosphine (TPP)
and diisopropylethylamine (DIPEA) at 60°C to obtain ben-
zylated aziridine 23 in 28% yield over two steps. Aziridine 2
was obtained after deprotection under Birch conditions (sodium
and t-butanol) with an overall yield of 11% from epoxide 16.
To synthesize ABPs, aziridine 23 was alkylated with 8-azidooc-
tyl triflate. Following Birch deprotection, amino-octylaziridine
3 was obtained in 54% yield over two steps. Aziridine 3 was
then coupled with either Cy5-OSu or biotin-OSu esters in the
presence of DIPEA to afford ABPs 4 and 5 following HPLC
purification.
Scheme 1: Synthesis of L-arabinofuranose-configured cyclopentene 15. Rea-
gents and conditions: a) (1S)-(+)-10-Camphorsulfonic acid, CH3CN, 50°C, 300
mbar, 2.5 h; b) BnBr, NaH, TBAI, DMF, 0°C, rt, 18 h, 74% over two steps; c)
BH3∙THF, Bu2BOTf, DMF, 0°C, 15 min, 90%; d) I2, TPP, THF, reflux, 3 h, 79%;
e) Activated Zn powder, THF, 35°C, 2 h, 84%; f) Ph3PCH3Br, n-BuLi, THF, -
78°C to -20°C for 1 h, then rt, 18 h, 73%; g) Grubb’s II cat., DCM, reflux, 18 h,
90%; h) DDQ, DCM, 0°C, rt, 2 h, 86%; i) Bu3SnMeI, KH, dibenzo-18-crown-6,
THF, 0°C, rt, 18 h, 91%; j) n-BuLi, THF, -78°C to rt, 18 h, 68%.
The first step towards the designed α-L-epoxide and α-L-aziri-
dine compounds was stereoselective epoxidation of cyclopen-
tene 15 (Scheme 2). We rationalized that treatment of cyclopen-
tene 15 with m-CPBA would lead to predominant β-L-epoxida-
tion where the neighboring primary alcohol would play a direct-
ing role by hydrogen bonding with m-CPBA. Indeed, m-CPBA
epoxidation at 50°C overnight resulted in a separable 3.4:1 mix-
ture of β-L- and α-L-epoxides in 62% yield. Cooling the mixture
to 4°C slowed the reaction, and after 4 days, we observed a β-L
to α-L ratio of 4.3:1, with a higher reaction yield (91%). To syn-
thesize the α-L-epoxide selectively, cyclopentene 15 was ben-
zylated and subjected to epoxidation with m-CPBA. Although
the β-L to α-L ratio was improved to 1:2, it resulted in a chro-
matographically inseparable mixture. Thus, α-L-arabino-
furanose-configured epoxide 1 was obtained by hydrogenation
of partially benzylated 17 with Pearson’s catalyst.
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AnAbfA (GH51)
1
2
3
4
5
6
7
8
KI
(M)
n.d.
140±20
210±30
n.d.
kinact
kinact/KI
(s-1M-1)
<0.1
39
7.1
Compound
(min-1)
n.d.
1
2
3
6
0.33±0.02
0.09±0.01
n.d.
160±20
AkAbfB (GH54)
n.d.
n.d.
<0.1
28
6.2
1
2
3
6
320±50
320±40
n.d.
0.54±0.07
0.12±0.01
n.d.
9
10
11
12
13
240±30
Table 1: Kinetic parameters for covalent inhibition of AnAbfA and AkAbfB by
α-L-arabinofuranosidase compounds 1, 2, 3, and 6. For reactions with
compound 6, it was not possible to obtain distinct kinact and KI parameters; only
the combined kinact/KI parameter determined from the slope of the kapp vs [I]
curve is shown for these cases. n.d.: not determined.
Scheme 3: Synthesis of α-L-aziridines 2-5. Reagents and conditions: a) BnBr,
14
15
16
17
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60
NaH, TBAI, DMF, rt, 18 h, 78%. b) NaN3, LiClO4, DMF, 100°C, 18 h 77%. c)
TsCl, DMAP, TEA, DCM, 0°C, 18 h, 50%. d) TPP, DIPEA, THF:H2O, reflux,
1.5 h, 56%. e) Li, NH3, -60°C, 1 h, 66%. f) 8-azidooctyl triflate, DIPEA, DCM,
0°C to rt, 18 h, 57%. g) Na, NH3, t-BuOH, -60°C, 1 h, 95%. h) Cy5-Osu or
biotin-OSu, DIPEA, DMF, 18 h, 4: 56% and 5: 19%.
As predicted by our conformational analysis, compound 6 is a
potent inhibitor of retaining α-L-arabinofuranosidases. Inhibitor
6 reacted rapidly with the catalytic nucleophile of both AkAbfB
and AnAbfA with a kinact well above 1 min-1 (estimated from the
limited speed of our assay). However, the lack of any apparent
non-linearity in the kapp vs. [I] curve for either AkAbfB or
AnAbfA suggested poor initial binding (Supplemental Figure
1Error! Reference source not found., Supplemental Figure
2Error! Reference source not found.). In spite of this, inhibi-
tor 6 has a performance constant of 170 M-1s-1 with AnAbfA
and 250 M-1s-1 with AkAbfB (Table 1), comparable to the inhi-
bition of TmGH1 with cyclophellitol reported by Gloster et al.11
(290 M-1s-1).
The synthesis of irreversible α-L-arabinofuranose configured
cyclic sulfate 6 (Scheme 4) started with the oxidation of 18 with
a mixture of NaIO4 and RuCl3∙3H2O affording exclusively cis-
α-L-diol 25 in 48% yield. Diol 25 was then treated with thionyl
chloride and trimethylamine, and the sulfite mixture was then
further oxidized with NaIO4 and RuCl3∙3H2O to give cyclic sul-
fate 26. This was deprotected using Pearson’s catalyst to afford
final cyclic sulfate 6 in 24% yield from 18.
Contrary to our prediction, compound 2 also proved to be a po-
tent inhibitor of both AkAbfB and AnAbfA, having perfor-
mance constants only 8-fold and 4-fold lower than inhibitor 6
with AkAbfB and AnAbfA, respectively (Table 1). In contrast
to inhibitor 6, inhibition kinetics with inhibitor 2 provided evi-
dence of stronger initial binding in both enzyme active sites,
having KI values of 0.1-0.3 mM. The addition of an alkyl chain
to generate inhibitor 3 did not hinder initial binding with either
AnAbfA or AkAbfB and caused only a 4-fold reduction in kinact
demonstrating that alkylation of the aziridine is a well-tolerated
method for generating α-L-arabinofuranosidase ABPs.
,
Scheme 4: Reagents and conditions towards cyclic sulfate 3. a) NaIO4, RuCl3∙3
H2O, EtOAc/CH3CN/H2O, 0°C, 3 h, 48%. b) i) SOCl2, Et3N, DCM, 0°C, 30 min.
ii) NaIO4, RuCl3∙3H2O, EtOAc/CH3CN/H2O, 0°C, 3 h, 51% c) H2, Pd(OH)2,
MeOH, 18 h, 24%.
The lack of measurable inhibition kinetics for compound 1 al-
lowed us to establish a maximum value for the putative inhibi-
tor’s performance constant (kinact/KI) of approximately 0.1 M-1s-
1 based on the length and sensitivity of the assay and the maxi-
mum inhibitor concentration tested (Supplemental Figure
1Error! Reference source not found., Supplemental Figure
2Error! Reference source not found.). Similarly, no reversi-
ble inhibition was observed at concentrations as high as 0.25
mM. Together, these results confirmed that, as predicted from
our conformational analysis, compound 1 was not an inhibitor
of retaining α-L-arabinofuranosidases at concentrations up to
0.25 mM.
Inhibition of recombinant α-L-arabinofuranosidases
With 1, 2, and 6 in hand, we first assessed the potency of these
putative inhibitors against their intended targets. To test the ef-
fectiveness of each inhibitor, inhibition kinetics were measured
with a collection of recombinantly produced retaining α-L-arab-
inofuranosidases including Geobacillus stearothermophilus
GH51 (GsGH51, a bacterial enzyme from GH51), Aspergillus
niger AbfA (AnAbfA, a fungal enzyme from GH51), and As-
pergillus kawachii AbfB (AkAbfB, a fungal enzyme from
GH54). Initial overnight incubations of each enzyme with com-
pounds 2 and 6 resulted in the complete loss of activity, while
no loss of activity was observed with compound 1. Intact MS of
GsGH51 and AkAbfB treated with each compound confirmed
complete 1:1 labelling with compounds 2 and 6, and no label-
ling with compound 1 (Supplemental Figure 7, Supplemental
Figure 8).
Structural analysis of inhibitors 2 and 6 bound to α-L-arab-
inofuranosidases
To determine whether inhibitors 2 and 6 both interact with α-L-
arabinofuranosidases as effective α-L-arabinofuranose mimics,
we sought to understand how the inhibitors bind to the enzyme
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active site. Soaking GsGH51 with inhibitors 2 and 6 overnight the binding energy of 6. Binding energy was less favorable with
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Figure 3. Crystal structures of complexes between inhibitors 2 (B, D, green) and 6 (A, C, purple), and GsGH51 (A, B, blue) and AkAbfB (C, D,
yellow). 2Fo-Fc electron density is shown for both the ligand and the catalytic nucleophile as a grey mesh contoured at 2σ. The polypeptide is shown
in cartoon form with active site residues shown as sticks. Apparent hydrogen bonding interactions are shown as dotted yellow lines.
at room temperature resulted in the formation of full occupancy
covalent complex between E294, the known catalytic nucleo-
phile, and each inhibitor (Figure 3A,B). These structures are
similar to the complexes reported by Hövel et al.57 They re-
ported a covalent substrate-enzyme intermediate trapped in a 2E
conformation (PDB ID 1PZ2); however, poor electron density
at the anomeric center of the Hövel structure limits the confi-
dence with which this ligand conformation can be interpreted.
Nevertheless, it is clear that the structure of the active site varies
between the Hövel complexes and the complexes with inhibi-
tors 2 and 6 that we obtained (Supplemental Figure 9).
either mutant (+4 kcal/mol for E175A and +8 kcal/mol for
E175G). We interpret this as indicating that the displacement of
E175 likely occurs following the reaction of the cyclic sulfate
with the covalent nucleophile. We speculate that the observed
active site rearrangement occurs following the addition reaction
and is not relevant to initial inhibitor binding. Overall, while
inhibitors 2 and 6 both bind in a manner mimicking the cognate
substrate of GsGH51, their labelling of the catalytic nucleophile
appears to induce significant rearrangement of the active site
structure.
Interestingly, following reaction with the catalytic nucleophile,
the conformation of inhibitor 2, but not inhibitor 6, appears to
represent the glycone conformation expected of the glycosyl en-
zyme intermediate. Reacted inhibitor 2 was found in the 2E con-
formation, forming hydrogen bonds from O2 to N174, from O3
to N74 and E29, and from O5 to Q351 and Y246. Inhibitor 6
formed the same complement of ligand-protein interactions but
sat in the active site in an unusual E1 conformation. We attribute
this to a combination of electrostatic repulsion and steric bulk
pushing the sulfate group out of the active site, promoting an
extended conformation for the bonds connecting Oε1 of E294
to the sulfate group.
Complexes with both inhibitors 2 and 6 are characterized by the
positioning of the E294 side chain away from R69, towards the
unliganded (PDBID: 1PZ3) position of Y246, which, in place
of a hydrogen bond to the ring oxygen of α-L-arabinofuranose,
forms a hydrogen bond with O5 of the inhibitor (carbohydrate
numbering). While the resulting displacement of Y246 has only
a minimal impact on the protein structure when bound to inhib-
itor 6, binding to inhibitor 2 results in the dramatic displacement
of Y246, and consequently W298 and N302, creating sufficient
space for the sidechains of I356 and L318 to pack into a differ-
ent position and for a glycerol molecule to bind. The presence
of the bulky charged sulfate group following reaction with in-
hibitor 6 appears to repel E175, the general acid/base residue,
displacing H244 and, through steric interactions, the S215-
R218 loop. To investigate whether E175 is interfering with the
binding of inhibitor 6, we simulated the Michaelis complex of
inhibitor 6 with the E175A and E175G mutants, and calculated
To generate covalent GH54 complexes, we soaked crystals of
AkAbfB with 0.2 mM of inhibitor 2 or 6 for 1 hour. Both inhib-
itors bound to E221 in almost identical positions and confor-
mations (Figure 3C,D), forming hydrogen bonds from O2 to
G296 and the sulfur of M195, from O3 to N297 and D219, and
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This is supported by the trends observed in the binding energies
from O5 to D219 and N223. The interaction between the ring
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oxygen and N222 found in the product complex (PDBID:
1WD4) cannot be formed, but the axial amine presents an addi-
tional hydrogen bond with D297, the general acid/base. In con-
trast to the complex with GsGH51, the interactions between
both inhibitors 2 and 6 and the active site of AkAbfB cause no
significant change in the protein structure. The active site ap-
pears to be sufficiently open to accommodate the sulfate of in-
hibitor 6 without any steric clashes. Thus, we believe that these
complexes are good representations of the glycosyl-enzyme in-
termediate structure. The ring in each covalent complex is
found in the 2E conformation. This consensus conformation rep-
resents a 1.2 Å migration of C1 from its position in the AkAbfB
Michaelis complex toward E221 coupled with a ~15° axial ro-
tation of the ring around C3 (Supplemental Figure 10).
calculated for the modelled Michaelis complexes of inhibitor 1,
2, and 6 in the GH51 and GH54 active sites (Supplemental Fig-
ure 12, Supplemental Figure 13). The binding energy of inhibi-
tor 2 was calculated in 3 different situations in each active site:
deprotonated inhibitor 2 with protonated acid/base residue, pro-
tonated inhibitor 2 with protonated acid/base residue, and pro-
tonated inhibitor 2 with deprotonated acid/base residue. Proto-
nated compound 2 (with the optimal acid/base residue protona-
tion) binds better than all the other compounds in both enzymes
(Supplemental Figure 14Error! Reference source not found.);
thus, not requiring the donation of a proton from the general
acid/base residue for the reaction to take place. Also, the im-
portance of the protonation state of the acid/base residue seems
to be different in both enzymes. GH54 with an extended acidic
range of labelling efficiency seems to have similar binding en-
ergies with either the protonated or deprotonated general
acid/base; whereas in GH51, 2 binds much better when it is
deprotonated, explaining the more restricted pH range of label-
ling efficiency.
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Probing the Aspergillus niger arabinan secretomes with
ABP 4
Building on the success of inhibitors 2 and 3 as covalent inhib-
itors of both GH51 and GH54 α-L-arabinofuranosidases, we set
out to detect and identify α-L-arabinofuranosidases within com-
plex fungal secretomes. As a validation of this approach, we
chose to work with the well-studied secretome of A. niger
grown on arabinan.
To investigate the thermal stability of arabinofuranosidases
within the A. niger arabinan secretome, we pre-incubated the
secretome at various temperatures for one hour prior to visuali-
zation with ABP 4. This revealed enzyme recovery from sur-
prisingly high temperature treatments (Supplemental Figure
15A,C). Both enzymes were stable up to 60°C. Increasing the
temperature to 65°C resulting in a complete loss of GH54 stain-
ing and raising it to 67°C resulted in a complete loss of both
GH51 and GH54 labeling, suggesting complete denaturation.
However, increasing the pre-incubation temperature beyond
67°C resulted in a partial recovery of GH54 staining. Pre-incu-
bation at 86.5°C resulted in a ~50% recovery of fluorescence
intensity relative to the RT control (estimated by band integra-
tion using ImageQuant software (GE)). To determine the role
of disulfide bonding in the stability AbfA and AbfB and the ap-
parent refolding of AbfB, we repeated the experiment with 5
mM DTT (Supplemental Figure 15B,C). The addition of DTT
had minimal impact on the apparent stability of AbfA, yet sig-
nificantly reduced the apparent stability of AbfB, causing a near
complete loss of staining at 56°C. This suggests that the four
disulfide bonds found in the structure of AkAbfB (conserved in
AnAbfB) are critical for enzyme stability, but that disulfide
bonds are not important for AbfA stability. Enzyme recovery
from elevated temperatures was reduced, but still occurred in
spite of the reduction of disulfide bonds.
Multiple α-L-arabinofuranosidases have been purified from the
A. niger secretome and characterized.62–64 These include
AnAbfA, the fungal GH51 that we produced recombinantly,
and AnAbfB, a GH54 enzyme 98% identical to AkAbfB. Thus,
we hypothesized that our ABPs could be used to identify the α-
L-arabinofuranosidases that are produced by A. niger in re-
sponse to a specific carbon source.
The treatment of the A. niger arabinan secretome with inhibitor
2 resulted in the complete loss of activity against 4MU-Araf,
suggesting that all of the α-L-arabinofuranosidase activity in our
secretome sample could be attributed to retaining glycosidases.
Visualization of α-L-arabinofuranosidases using ABP 4 re-
vealed two distinct bands, one running at ~105 kDa and the
other running at ~65 kDa (Figure 4A). Deglycosylation with
PNGaseF under denaturing conditions resulted in a shift of the
105 kDa band down to ~70 kDa and a shift of the ~65 kDa band
down to ~60 kDa. Based on similar results obtained with re-
combinant AnAbfA and AkAbfB, we hypothesized that the top
band was one of the A. niger GH51 enzymes and the bottom
band was AnAbfB, the only A. niger GH54 enzyme.
To determine whether the recovery of AbfB staining was genu-
inely related to the recovery of active enzyme, we measured hy-
drolytic activity of the DTT treated secretome samples towards
4MU-Araf (Supplemental Figure 15D). This confirmed that the
loss of AbfB staining at 56°C correlated with an ~80% reduc-
tion in activity and that the subsequent loss of AbfA staining at
67°C corresponded with a complete loss of activity. At higher
temperatures we observed a small recovery of activity which
correlated with the recovery of AbfB staining. Thus, visualiza-
tion with ABP 4 facilitates the identification of thermally resil-
ient enzymes within the context of their native fungal secre-
tome.
Investigations of the effects of pH on labelling efficiency re-
vealed that AnAbfB reacted with our probe efficiently over a
pH range (2-9), which extended further into the acidic range
than the GH51 enzyme (5-8) (Supplemental Figure 11). Nota-
bly, both enzymes were labelled optimally at pH 6.5-7, which
is significantly above pH 4, at which the enzymes are optimally
active. While similar discrepancies between optimal hydrolytic
and inhibition pH have been reported previously15,18, the differ-
ence of 2.5-3 pH units that we observed is unusually large, sug-
gesting that the optimal protonation states of active site residues
for inhibition by compound 2 and glycoside hydrolysis are dif-
ferent.
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Figure 4. Activity-based protein profiling of fungal secretomes with ABPs 4 and 5. A) Fluorescence imaging of the secretome isolated from A. niger grown on
arabinan, stained with ABP 4, and treated with (PNG+) or without (PNG-) PNGaseF under denaturing conditions prior to separation on an 8.75% SDS-PAGE gel. L
indicates the ladder lanes. B) Label-free quantification of the top eight proteins pulled down from the A. niger arabinan secretome. For each protein (identified by
NRRL3 number and common name), integrated peptide intensity is plotted for non-conflicting peptides from the pull-down with ABP 5 (PD, black), from the total
secretome (TS, blue), and from the pull-down with ABP 5 following pre-treatment with inhibitor 2 (PT, red). Error bars represent the standard deviations of three
measurements. C) Cy5 fluorescence (red) and Coomassie staining (green) of basidiomycete secretomes following staining with ABP 4 and acetone precipitation. L
indicates the ladder lane. The BRFM number for the strain from which the secretome was isolated is given above each lane. D) Plot of total spectral counts in the
pull-down sample vs. the ratio of spectral counts in the pull-down sample to spectral counts in the total secretome for all of the proteins for which at least 2 peptides
were observed with an FDR of 1% in the pull-downs from L. menziesii (BRFM 1557), F. fomentarius (BRFM 1323), T. gibbosa (BRFM 952), and A. biennis (BRFM
1215). Points corresponding to GH51 enzymes are shown in orange, points corresponding to other putative retaining GH enzyme with peptide molecular weights
>90 kDa are shown in red. The labels shown include the species abbreviation and the Mycocosm amino acid sequence number.
arabinan-rich sugar beet pectin62. The genome of A. niger en-
Identification of A. niger α-L-arabinofuranosidases by pull-
codes two other GH51 genes: abfD, which is not expressed dur-
down with ABP 5
ing growth on arabinan, and the more recently identified abfE,
Based on molecular weight and glycosylation state, we hypoth-
for which expression has not been investigated in response to
esized that the enzymes stained by ABP 4 were a GH51 and a
arabinan.
GH54. However, it was not clear which of the GH51 enzymes
produced by A. niger was expressed. A previous report has
identified AbfA, AbfB, and AbfC as the major α-L-arabino-
furanosidases produced by A. niger in response to growth on
On-bead digestion of proteins pulled down following treatment
of the secretome with ABP 5 yielded peptides from AbfB
(GH54, GenBank: CAK42333), AbfA (GH51, GenBank:
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Bands stained with ABP 4 in these samples were identified by
CAK43424), and AbfE (GH51, GenBank: ACE00420) as well
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5
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8
pull-down using the same protocol as for the A. niger secre-
tome, but with an added 10-minute wash of the beads with 2%
SDS at 65°C to more strictly eliminate any proteins non-specif-
ically bound to the beads. Comparing proteomic analyses of the
secretome and pull-down samples, the vast majority of proteins
found within the secretome were rendered undetectable by our
washing protocol. In spite of this, the proteins confidently ob-
served (at least two unique peptides identified with an FDR of
1%) were still predominantly not enzymes phylogenetically re-
lated to known α-L-arabinofuranosidases. Considering the
abundance of GH7 enzymes apparent in the total secretome and
in the pull-down, and the apparent staining of the same enzymes
by ABP 4 (~50-60 kDa bands in Figure 4C), we believe that
many of these hits represent non-specific labelling of abundant
species within the secretome (e.g. GH7 enzymes Lmen|932922,
Ffom|431808, and Tgib|1002594).
as a small collection of other proteins not known to be α-L-arab-
inofuranosidases (Figure 4B). We did not observe AbfC or
AbfD in our analysis of the pull-down total secretome, indicat-
ing that these were not produced in our culture. Pre-incubation
of the secretome with inhibitor 2 followed by treatment with
ABP 5 and pull-down significantly reduced signal for peptides
from AbfA, AbfB, and AbfE without causing a significant re-
duction in signal for any other detected proteins. Although we
cannot exclude that ABP 5 has specific targets beyond arabino-
furanosidases that are incapacitated by inhibitor 2, these results
reveal the utility of ABP 5 in activity-based protein profiling to
identify and annotate retaining arabinofuranosidases from se-
cretomes derived from microorganisms grown on arabino-
furanose-containing biopolymers.
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Screening basidiomycetes for α-L-arabinofuranosidase
production
Thus, we combined the metrics of total spectral counts from the
pull-down (SC(PD), a rough measure of abundance) and the ra-
tio of spectral counts from the pull-down to spectral counts from
the digestions of the total secretome (SC(PD)/SC(TS), a rough
measure of selectivity) to give the plot shown in Figure 4D.
Three GH51 enzymes (JGI ProtIDs Lmen|915930,
Tgib|1320025, and Ffom|1458192) appear as distinct targets of
ABP 5 with elevated SC(PD) and SC(PD)/SC(TS). These
GH51 enzymes had masses correlated with the masses of the
most intense bands observed by visualization with ABP 4 (those
between 70 and 100 kDa). A single GH51 enzyme in the same
mass range from A. biennis (Abien|540325) was detected in the
pull-down sample. However, the signal was weak, with only 4
spectral counts detected.
Following the success of the detection and identification of A.
niger α-L-arabinofuranosidases, we applied ABPs 4 and 5 to the
detection and identification of α-L-arabinofuranosidases se-
creted by basidiomycetes grown on complex biomass. We se-
lected a sampling of eight basidiomycetes, all known to be pro-
ficient biomass-degrading fungi (Supplemental Table 3). The
genomes of these fungi encode no apparent GH54 enzymes and
either one (A. biennis and T. gibbosa) or two apparent GH51
enzymes. To identify the GH51(s) produced during growth on
complex biomass, these fungi were cultured on maltose, aspen
pulp, or wheat straw for 10 days prior to secretome collection.
α-L-Arabinofuranosidases were visualized by treatment of se-
cretome samples with ABP 4 at pH 5.5, 30°C for 1 hour fol-
lowed by denaturation, deglycosylation, and separation on
SDS-PAGE. Glycoproteins migrating at 70-80 kDa were ob-
served in secretomes collected from T. gibbosa (the top biomass
digestion-enhancing strain identified in a sampling of French
biomass-degrading fungi65), F. fomentarius (a white-rot fungus
which grows on hardwood trees66), and L. menziesii and A. bi-
Based on the limited number of hits in the pull-down samples
with predicted molecular weights above 90 kDa, we believe that
the higher molecular weight bands (100-120 kDa) observed in
the SDS-PAGE of secretomes from T. gibbosa, A. biennis, and
F. fomentarius are GH3, GH31, or GH35 enzymes. Most of
these enzymes were observed with poor SC(PD) and
SC(PD)/SC(TS) values (red dots in Figure 4D), however a sin-
gle GH3 enzyme (Tgib|1466933) appeared to be a target of ABP
5. While this may represent substrate flexibility within these
GH families, it remains to be determined whether these en-
zymes display significant α-L-arabinofuranosidase activity.
ennis (both known to be effective in biomass pre-treatment67,68
)
when grown on aspen pulp (Supplemental Figure 16A). T. gib-
bosa and L. menziesii secretomes gave the same band following
growth on wheat straw while the secretomes of A. biennis and
F. fomentarius did not (Supplemental Figure 16B). T. gibbosa,
F. fomentarius, and A. biennis did not produce any apparent α-
L-arabinofuranosidase when grown on maltose. However, sur-
prisingly, L. menziesii did. Coomassie staining showed very lit-
tle total protein present in any of the secretome samples
(Supplemental Figure 16C, D), demonstrating the remarkable
sensitivity of ABP 4.
CONCLUSIONS
The discovery of the mechanism-based covalent α-L-arabino-
furanosidase inhibitors 2 and 6 expand the library of tools available
for the characterization of enzymes expressed during plant biomass
degradation. The unexpectedly high efficiency of inhibitors 2 and
3 provided a platform on which ABPs for α-L-arabinofuranosidases
could be synthesized. The potential of ABPs 4 and 5 in the discov-
ery, identification, and characterization of α-L-arabino-
furanosidases from fungal secretomes grown on both arabinose-
rich biomass and complex woody biomass has been demonstrated.
We envision that the ability to efficiently screen samples of interest
for levels of multiple active α-L-arabinofuranosidases will facilitate
and accelerate a variety of applications including enzyme discov-
ery, bioprocess monitoring, and the investigation of plant-pathogen
interactions.
Based on these results, T. gibbosa, L. menziesii, A. biennis, and
F. fomentarius were selected for follow-up studies. Staining
100 μL of secretome followed by acetone precipitation gave
much higher band intensity compared to the effective loading
of ~2.8 µL in the screening experiment. This revealed a collec-
tion of 1-3 bands running between 55 and 130 kDa (Figure 4C,
and Supplemental Figure 17). Coomassie staining of the same
gel revealed a broad range of bands, few of which appeared to
co-migrate with the bands detected by visualization with ABP
4 (Figure 4C).
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BRFM 1802, Trametes ljubarskyi BRFM 1659, Leiotrametes men-
ziesii BRFM 1781, Fomes fomentarius BRFM 1823 and Trametes
meyenii BRFM 1810.
ASSOCIATED CONTENT
Supporting information for this article includes:
-Supplemental figures and table
1
2
3
4
5
6
7
8
9
-Supplemental synthetic methods and characterization for com-
pounds 1-23
This material is available free of charge via the Internet at
ABBREVIATIONS
4MU-Araf: 4-methylumbelliferyl α-L-arabinofuranoside
a.u.: Atomic units
ABP: Activity-based probe
CP: Car-Parrinello
DDQ: 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone
DIPEA: Diisopropylethylamine
DPH: O-(2,4-dinitrophenyl)hydroxylamine
FEL: Free-energy landscape
GH51: Glycoside hydrolase family 51
GH54: Glycoside hydrolase family 54
MD: Molecular dynamics
http://pubs.acs.org
Crystallographic structural models and diffraction data can be
accessed at www.rcsb.org under the PDB IDs 6SXV, 6SXU,
6SXT, 6SXS, and 6SXR.
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Proteomic LC-MS/MS datasets have been deposited with the
MassIVE database (massive.ucsd.edu, UCSD) with the identi-
fiers MSV000084886 (A. niger arabinan secretome) and
MSV000084877 (Basidiomycete secretomes) and can be ac-
cessed via FTP.
PD: Pull-down
PIDA: Phenyliodine(III) diacetate
PNP-Araf: 4-nitrophenyl α-L-arabinofuranoside
PMB: Para-methoxybenzyl
PMP: Para-methoxybenzylidene
RCM: Ring-closing metathesis
SC: Spectral counts
SD: Standard deviation
SPG: Succinate-phosphate-glycine
TPP: Triphenylphosphine
TS: Total secretome
AUTHOR INFORMATION
Corresponding Author
*Correspondence should be addressed to HSO <h.s.overk-
leeft@lic.leidenuniv.nl>, CR <c.rovira@ub.edu> or GJD <gid-
eon.davies@york.ac.uk>
Funding Sources
We thank the Natural Sciences and Engineering Research Council
of Canada (Post-Doctoral Fellowship to NGSM), the Royal Society
(Ken Murray Research Professorship to GJD), the Biotechnology
and Biological Sciences Research Council (BBSRC) (grant
BB/R001162/1 to GJD), the Netherlands Organization for Scien-
tific Research (NWO TOP grant 2018-714.018.002 to HSO), the
European Research Council (ERC-2011-AdG-290836 “Chembio-
sphing” to HSO), the Generalitat de Catalunya (FI-AGAUR PhD
scholarship to AN-H), the computer resources at MareNostrum IV
and MinoTauro and the technical support provided by the Barce-
lona Supercomputing Center (BCV-2016-2-0002 and BCV-2016-
3-0005), and the computational support from the University of
York High Performance Computing service, Viking and the Re-
search Computing team (chem-menz-2019). Joint Genome Insti-
tute, a DOE Office of Science User Facility, is supported by the
Office of Science of the U.S. Department of Energy under Contract
No. DE-AC02-05CH11231. The authors declare no competing fi-
nancial interest.
Q-CF3: 3-amino-2-(trifluoromethyl)quinazolin-4(3H)-one
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ACKNOWLEDGMENT
The authors would also like to thank professors Takuya Koseki and
Shinya Fushinobu for providing the expression plasmid for
AkAbfB. The authors would like to thank Diamond Light Source
for beam time (proposal 18598), and the staff of beamline I04 for
assistance with crystal testing and data collection. Proteomics data
were collected at the York Centre of Excellence in Mass Spectrom-
etry, which was created thanks to a major capital investment
through Science City York, supported by Yorkshire Forward with
funds from the Northern Way Initiative, and subsequent support
from EPSRC (EP/K039660/1; EP/M028127/1). Wiley-milled as-
pen (Populus grandidentata) was kindly provided by Dan Cullen
(Forest Product Laboratory, USDA, Madison, WI, USA). The au-
thors are grateful to Igor V Grigoriev, Joint Genome Institute for
providing access to genome sequence data for Trametes gibbosa
BRFM 1770, Abortiporus biennis BRFM 1778, Hexagonia nitida
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SUPPLEMENTAL FIGURE AND TABLE CAPTIONS
Supplemental Figure 1. Inhibition kinetics of AnAbfA. A) Plot of rate vs. substrate concentration measured for AnAbfA hydrolyzing 4MU-Araf with the hyperbolic fit
shown as a dotted line. B) Plot of fluorescence vs. time for AnAbfA in the presence of difference concentrations of inhibitor 1. Each line represents the average of
four measurements with the standard deviation represented as thinner lines above and below the average. C) Plot of fluorescence vs. time for AnAbfA in the
presence of difference concentrations of inhibitor 6 as shown in panel B. D) Plot of apparent decay constant (kapp) extracted from an exponential decay fit of the
curves shown in panel C vs. inhibitor concentration with the hyperbolic fit shown as a dotted line. Error bars are the standard deviation of the kapp values extracted
from each of the four measured curves. Panels E and F are the same measurements as panels C and D, but made with inhibitor 2. Panels G and H are the same
measurements as panels C and D, but made with inhibitor 6.
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Supplemental Figure 2. Inhibition kinetics of AkAbfB. A) Plot of rate vs. substrate concentration measured for AkAbfB hydrolyzing 4MU-Araf with the hyperbolic fit
shown as a dotted line. B) Plot of fluorescence vs. time for AkAbfB in the presence of difference concentrations of inhibitor 1. Each line represents the average of
four measurements with the standard deviation represented as thinner lines above and below the average. C) Plot of fluorescence vs. time for AkAbfB in the
presence of difference concentrations of inhibitor 6 as shown in panel B. D) Plot of apparent decay constant (kapp) extracted from an exponential decay fit of the
curves shown in panel C vs. inhibitor concentration with the hyperbolic fit shown as a dotted line. Error bars are the standard deviation of the kapp values extracted
from each of the four measured curves. Panels E and F are the same measurements as panels C and D, but made with inhibitor 2. Panels G and H are the same
measurements as panels C and D, but made with inhibitor 6.
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Supplemental Figure 3: Determination of the impact of inner filter effects and stability of fluorescence over the course of the inhibition measurement. A) Epifluores-
cence intensity vs. [4MU] measured in 40 µL total volume of pH 7 phosphate buffer in a black 384-well plate using different excitation wavelengths. A linear best fit
for F vs. [4MU] from 0.125 to 100 µM at 360 nm excitation is shown as a dashed blue line. A linear best fit for F vs. [4MU] from 0.125 to 500 µM at 390 nm excitation
is shown as a dashed purple line. B) 4MU epifluorescence intensity measured over 200 minutes with a 15 second cycle time using an excitation wavelength of 360
nm at 25°C for concentrations ranging from 0.125-100 µM in 40 µL of pH 7 phosphate buffer in a black 384-well plate, plotted on a log scale.
Supplemental Figure 4: Comparison of inhibitor pre-treatment efficiency for blocking α-L-arabinofuranosidase active sites. To identify conditions suitable for blocking
α-L-arabinofuranosidase active sites prior to probe labelling, pretreatments with 0.8-100 µM of inhibitors 1, 2, or 6 were performed followed by staining with probe
4. Each pretreatment and staining was run in duplicate and were loaded into the gel in replicate pairs. The compound number and concentration are given above
each pair of lanes.
Supplemental Figure 5. A) Crystals of GsGH51 grown at 293 K from a mixture of 1200 nL of 8 mg/mL GsGH51 in 5 mM Tris-HCl, 1 mM EDTA, pH 8 mixed with 600
nL of 0.1 M Tris-HCl, pH 7.5, 5% 2-propanol, 0.7 M NH4F, 20% PEG3350. B) Crystals of EndoH-deglycosylated AkAbfB grown at 293 K from a mixture of 1200 nL
of 10 mg/mL AkAbfB in 20 mM pH 5.0 sodium acetate mixed with 600 nL of 50% PEG400, 0.4 M Li2SO4, 100 mM sodium acetate, pH 4.5.
Supplemental Figure 6. Structure of the AkAbfB (E221Q) PNP-Araf Michaelis complex. A) Electron density of the PNP-Araf molecule (blue) within the AkAbfB
(E221Q) active site (yellow) contoured to 1σ. Apparent hydrogen bonding interactions are shown as yellow dotted lines. B) Superposition of the AkAbfB (E221Q)
Michaelis complex (yellow) with the AkAbfB product complex (fuchsia, PDBID: 1WD4).
Supplemental Figure 7. Deconvoluted intact MS measurements of GsGH51 before and after treatment with inhibitors 1 (A), 2 (B), and 6 (C). Predicted mass =
57406.3, expected Δm (1) = 146, expected Δm (2) = 145, expected Δm (6) = 226
Supplemental Figure 8. Deconvoluted intact MS measurements of AkAbfB before and after treatment with inhibitors 2 and 6. The predicted mass is unknown due
to unknown glycosylation remaining after EndoH treatment. Expected Δm (2) = 145, expected Δm (6) = 226. Note the additional peak series in the 6-treated sample,
which corresponds to a loss of 80 Da, the mass of SO3. We attribute this to in-source fragmentation.
Supplemental Figure 9. Perturbation of the GsGH51 active site following reaction with inhibitor 2. The complex with inhibitor 2 is shown in blue, the complex with
inhibitor 6 is shown in cyan, and the complex with arabinofuranose (PDBID: 1PZ2) is shown in fuchsia. With the exception of Asn74 and Trp99 (shown for reference),
the ligand and the active site residues with significantly different positions in complex with inhibitor 2 are shown as sticks.
Supplemental Figure 10. Overlay of PNP-Araf and inhibitor 2 in the active site of AkAbfB (E221Q) (gold) and AkAbfB (green), respectively, showing the displacement
of the C1, C2, and O (or C6) positions between initial binding and the formation of the covalent intermediate.
Supplemental Figure 11. Fluorescence image (Cy5) following SDS-PAGE separation of A. niger arabinan secretome treated with ABP 4 in buffers at different pH
values. A) Three separate gels, which were imaged in parallel, are shown, separated by black lines. The buffer used during treatment with ABP 4 is labelled above
each lane (McI for McIlvane buffer and SPG for SPG buffer). B) Plot of normalized integrated band intensity vs. pH for both the upper (GH51) and lower (GH54)
bands in the McI and SPG buffer systems.
Supplemental Figure 12. Simulated optimized Michaelis complex configurations of inhibitors 1 (A), 2 (B, C, D), and 6 (E) used for the calculation of binding energy
within the active site of GsGH51. B shows the protonated general acid/base residue with deprotonated aziridine nitrogen, C shows the protonated general acid/base
residue with protonated aziridine nitrogen, D shows the deprotonated general acid/base residue with protonated aziridine nitrogen.
Supplemental Figure 13. Simulated optimized Michaelis complex configurations of inhibitors 1 (A), 2 (B, C, D), and 6 (E) used for the calculation of binding energy
within the active site of AkAbfB. B shows the protonated general acid/base residue with deprotonated aziridine nitrogen, C shows the protonated general acid/base
residue with protonated aziridine nitrogen, D shows the deprotonated general acid/base residue with protonated aziridine nitrogen,
Supplemental Figure 14: Relative energies of the inhibitors binding to the active sites of GsGH51 and AkAbfB. Compounds 2B, 2C and 2D corresponds to the
different tested protonation states, following the labels used in Supplemental Figure 10 and 11. 2B corresponds to protonated general acid/base with deprotonated
aziridine nitrogen of 2; 2C corresponds to protonated general acid/base with protonated aziridine nitrogen of 2 and 2D corresponds to deprotonated general acid/base
with protonated aziridine nitrogen of 2.
Supplemental Figure 15. A) Fluorescence image (Cy5) following SDS-PAGE separation of A. niger arabinan secretome treated with ABP 4 following incubation at
different temperature values. The incubation temperature prior to labelling with ABP 4 is written above each lane in °C. B) and C) Replication of the experiment
shown in A, but with the addition (B) or not (C) of 5 mM DTT prior to incubation at temperature. D) Plot of normalized integrated fluorescence intensity for the AbfA
and AbfB bands vs. pre-treatment temperature. E) Plot of absolute integrated fluorescence intensity for the AbfA and AbfB bands across the same range of temper-
ature with and without 5 mM DTT. The black line represents integrations from the gel in panel B and the red line represents integrations from the gel in panel C. F)
Plot of initial 4MU-Araf hydrolysis rate vs. pre-incubation temperature for 5 µL of arabinan secretome diluted into 45 µL of 50 µM 4MU-Araf in pH 6.5 phosphate
buffer. Each point represents the average of 4 technical replicates with error bars representing the standard deviation.
Supplemental Figure 16. SDS-PAGE of basidiomycete secretomes. A, B) Cy5 fluorescence scans of basidiomycete secretomes treated with ABP 4 and separated
on SDS-PAGE. Labels are shown above each lane. L indicates ladder, A indicates secretome grown on aspen pulp, W indicates secretome grown on wheat straw,
M indicates secretome grown on maltose, + indicates treatment with PNGaseF, - indicates no treatment with PNGaseF, the number above each lane is the BRFM
number for the strain from which the secretome was isolated. C, D) Coomassie staining of the same gels shown in A and B; labelling is identical.
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Supplemental Figure 17. Basidiomycete secretomes stained with ABP 4. Panel A is white light imaging following Coomassie staining, panel C is Cy5 fluorescence,
and panel B is Cy5 fluorescence (red) superimposed on the Coomassie image (image inverted, contrast optimized and false coloured green). Lane 1 contains 5 μL
of Pageruler 10-180 kDa prestained ladder. Lanes 2-5 are T. gibbosa, A. biennis, L. menziesii, and F. fomentarius, respectively.
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Supplemental Table 1: Kinetic parameters determined for the hydrolysis of 4MU-α-L-arabinofuranoside by AnAbfA and AkAbfB
Supplemental Table 2. Data collection and refinement statistics (molecular replacement).
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Supplemental Table 3: Basidiomycete strains screened for α-L-arabinofuranosidase production.
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