Evidence for a boat conformation at the transition state of GH76 α-1,6-mannanases - Key enzymes in bacterial and fungal mannoprotein metabolism

Article abstract of DOI:10.1002/anie.201410502

α-Mannosidases and α-mannanases have attracted attention for the insight they provide into nucleophilic substitution at the hindered anomeric center of α-mannosides, and the potential of mannosidase inhibitors as cellular probes and therapeutic agents. We

Full text of DOI:10.1002/anie.201410502

Angewandte
Chemie
DOI: 10.1002/anie.201410502
Conformational Analysis
Evidence for a Boat Conformation at the Transition State of GH76 a-
,6-Mannanases—Key Enzymes in Bacterial and Fungal Mannoprotein
1
Metabolism**
Andrew J. Thompson, Gaetano Speciale, Javier Iglesias-Fernꢀndez, Zalihe Hakki, Tyson Belz,
Alan Cartmell, Richard J. Spears, Emily Chandler, Max J. Temple, Judith Stepper,
Harry J. Gilbert, Carme Rovira,* Spencer J. Williams,* and Gideon J. Davies*
Abstract: a-Mannosidases and a-mannanases have attracted
attention for the insight they provide into nucleophilic
substitution at the hindered anomeric center of a-mannosides,
and the potential of mannosidase inhibitors as cellular probes
and therapeutic agents. We report the conformational itinerary
of the family GH76 a-mannanases studied through structural
analysis of the Michaelis complex and synthesis and evaluation
of novel aza/imino sugar inhibitors. A Michaelis complex in an
includes both mammalian and bacterial endo-a-mannosidases
and endo-a-mannanases; and GH76, featuring endo-acting
[3,5]
bacterial a-1,6-mannanases
and fungal transglycosi-
A growing body of literature has reported detailed
characterization of various GH99 endo-a-mannosidases in
[2,6]
dases.
[
4a]
[7]
[8]
terms of function, cellular localization, structure, and
the development of effective inhibitors that are active within
[9]
cells; by comparison our knowledge and understanding of
the biological role of GH76 enzymes has trailed. While
several GH76 structures are available, less is known of the
biochemistry and mechanism of these enzymes, no inhibitors
have been reported and nothing is known of the conforma-
tional changes occurring during catalysis.
O
S2 conformation, coupled with distortion of an azasugar in an
inhibitor complex to a high energy B2,5 conformation are
rationalized through ab initio QM/MM metadynamics that
show how the enzyme surface restricts the conformational
landscape of the substrate, rendering the B2,5 conformation the
most energetically stable on-enzyme. We conclude that GH76
enzymes perform catalysis using an itinerary that passes
Most a- and b-mannosidases perform catalysis through
one of two conformational itineraries. X-ray structures of
insightful ligand complexes, dovetailed with computational
analyses of conformational free-energy landscapes (FEL) of
inhibitors on- or off-enzyme, have allowed assignment of an
O
°
2,5
through S and B
conformations, information that should
2
inspire the development of new antifungal agents.
O
°
2,5
1
E
nzymes catalyzing the hydrolysis of glycosidic bonds within
S $B $ S itinerary to a- and b-mannosidases of families
2
5
[10]
a-mannoside-based glycans and glycoconjugates (a-manno-
sidases and a-mannanases) are of great interest due to their
GH2, 26, 38, 92, and 113.
Alternatively, an unusual
°
3
3
1
“southern hemisphere” itinerary, S ! H ! C , has been
1
4
4
[1]
[11]
roles in glycoprotein maturation, cell wall assembly in
fungi, and processing of complex dietary polysaccharides by
the human gut microbiota. Relatively little attention has
been directed at endo-acting a-mannosidases and a-manna-
nases, enzymes that play major roles in eukaryotic N-glycan
processing and in the metabolism and deconstruction of
fungal cell wall a-mannans.
grouped into two families within the CAZy sequence-
based classification (http://www.cazy.org): GH99, which
assigned to exo-acting a-mannosidases of family GH47.
[2]
Much effort has gone into the synthesis of enzyme inhibitors
that either report on, or reflect, these conformational path-
ways. We recently provided evidence that the mannosidase
inhibitor, mannoimidazole, is an exquisitely informative
conformational probe, owing to the relatively small energy
differences and barrier-less interconversion between ground-
[
3]
[
4]
[3]
Endo-a-mannanases are
[5]
4
3
2,5
state H or H conformations, and higher energy B or B2,5
3
4
[
10e]
conformations.
In contrast, the azasugar isofagomine
[*] Dr. A. J. Thompson, R. J. Spears, E. Chandler, Dr. J. Stepper,
M. J. Temple, Prof. H. J. Gilbert
Prof. G. J. Davies
Department of Chemistry, University of York
Heslington, York, YO10 5DD (UK)
Institute for Cell and Molecular Biosciences
The Medical School
Newcastle University, Newcastle upon Tyne, NE2 4HH (UK)
E-mail: gideon.davies@york.ac.uk
[
**] We thank the Australian Research Council, the UK Biotechnology
and Biological Sciences Research Council, Amicus Therapeutics, the
Spanish Ministry of Economy and Competitiveness, the Generalitat
de Catalunya, and the European Research Council. We also
acknowledge the staff of the Diamond Light Source (Didcot (UK))
for provision of beamline facilities, and the support, technical
expertise, and assistance provided by the Barcelona Supercomput-
ing Center: Centro Nacional de Supercomputaciꢄn. Supporting
information for this article is given via a link at the end of the
document.
G. Speciale, Z. Hakki, T. Belz, Dr. A. Cartmell, Prof. S. J. Williams
School of Chemistry and Bio21 Molecular Science and Biotech-
nology Institute, University of Melbourne
Parkville, Vic 3010 (Australia)
E-mail: sjwill@unimelb.edu.au
J. Iglesias-Fernꢀndez, Prof. C. Rovira
Departament de Quꢁmica Orgꢂnica and Institut de Quꢁmica Teꢃrica
i Computacional (IQTCUB), Universitat de Barcelona
08028 Barcelona (Spain)
E-mail: c.rovira@ub.edu
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201410502.
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(
IFG) was highlighted as a much poorer conformational
4
probe owing to a greater preference for the ground-state C1
conformation, and high-energy barriers that must be crossed
to attain mechanistically relevant half-chair or boat confor-
[10e]
mations.
In corollary, when a nonchair conformation is
observed for IFG-type inhibitors bound to a glycosidase, this
should be considered highly significant, with the conforma-
tional preferences of the enzyme overwhelming the otherwise
dominating intrinsic conformational bias of the inhibitor.
Here, we performed a detailed mechanistic, structural,
and conformational analysis of the catalytic domain of
Bacillus circulans Aman6 (herein termed BcGH76; see the
Supporting Information (SI) for details), the founding
Figure 1. Three-dimensional structure, sequence conservation, and
active site of GH76 a-1,6-mannanases. A) B. circulans TN-31 Aman6
catalytic domain (BcGH76) in complex with a-1,6-mannopentaose with
surface colored by sequence conservation using the partial GH76
alignment as shown in Figure S5. B) Complex with a-1,6-mannopen-
taose showing proposed catalytic nucleophile (Asp124) and general
acid/base variant (Asn125). C) Complex with ManIFG 3. Electron
density maps are REFMAC maximum-likelihood/s -weighted 2F ꢀF
[
12]
member of the GH76 family of a-mannanases.
Three-
dimensional (3D) structural analysis of the enzyme in diverse
complexes at near atomic resolution reveals details of the
catalytic conformational itinerary. Significantly, the Michaelis
A
o
c
(
enzyme-substrate) complex shows that the active center
3
syntheses contoured at 0.36 and 0.41 electrons per ꢅ , respectively.
Panel A was assembled using PyMOL v1.6 (Schrçdinger), panel B was
O
mannoside is distorted to an S2 skew-boat conformation
whilst a complex with the bespoke a-1,6-mannanase-targeted
inhibitor 1,6-ManIFG 3 shows mechanistically relevant dis-
tortion, with the piperidine ring adopting a high-energy B2,5
conformation. QM/MM metadynamics simulations show how
[17]
assembled using CCP4mg.
binding subsites (Figure 1; for subsite nomenclature see
Ref. [15]). Both 1 and 2 bound in similar positions (ꢀ3/ꢀ2)
away from the active center (see SI). In contrast, the
Michaelis complex with a-1,6-mannopentaose spans the
[
11b]
[10e]
the isolated mannoside
and IFG free-energy landscapes
are strongly perturbed on-enzyme, favoring BcGH76 catalysis
°
through a B2,5 transition state (TS ) conformation.
complete active center from ꢀ4 to + 1 and shows distortion
O
of the ꢀ1 subsite mannoside to an S conformation; highly
2
[
10b,c,16]
O
°
2,5
1
indicative
of a S $B $ S conformational pathway
2
5
for GH76 catalysis. 1,6-ManIFG 3 binds to BcGH76 with K =
d
1
.1 mm (see SI), some 5000 times stronger than the KM for
pNPMan , and occupies the (ꢀ2/ꢀ1) subsites. Notably, the
2
BcGH76-3 complex shows a distortion of the ꢀ1 moiety to
a B conformation (discussed below).
2
,5
[
12a]
Consistent with previous reports,
BcGH76 is inactive
The structures of the Michaelis and BcGH76-3 complexes
provide the first examples of a GH76 enzyme with ligands
bound at the ꢀ1 subsite. GH76 enzymes act with a net
against a-1,6-mannobiose (ManMan) and p-nitrophenyl a-
mannoside (pNPMan); however, kinetic parameters could be
[
3]
determined
using
p-nitrophenyl
a-1,6-mannobioside
retention of anomeric configuration, consistent with catal-
ysis using a classical Koshland double displacement mecha-
nism. In the BcGH76-3 complex the endocyclic nitrogen of
IFG (equivalent to the anomeric carbon) engages in a close
contact (2.8 ꢀ) with Asp124, congruous with other complexes
of IFG-type sugars with retaining glycosidases (see SI). In the
Michaelis complex Asp124 is poised for in-line nucleophilic
attack on the anomeric center of the substrate with Onuc–C1
distance 3.14 ꢀ and an Onuc-C1-OLG angle of 1608. The
neighboring amino acid, Asp125, occupies a position consis-
tent with that of an anti-protonating general acid/base residue
and is H-bonded to the leaving group glycosidic oxygen within
the Michaelis complex. These structure-based assignments
were investigated by mutagenesis. BcGH76 D124N showed
no activity against either pNPMan or a-1,6-Man , consistent
ꢀ
1
ꢀ1
(
(
pNPMan , k /K = 1.11 min mm ) and a-1,6-mannotriose
2
cat
M
ꢀ1
ꢀ1
a-1,6-Man , k /K = 27.7 min mm ; see SI for details).
3
cat
M
Unlike various exo-mannosidase families (compare GH
[
13]
families 38, 47, and 92), which employ divalent metal ions
to coordinate and distort the geometry of the substrate,
BcGH76 is metal-independent, with no change in enzymatic
activity in the presence of EDTA, and no evidence of metal
ion coordination in any structures determined.
The 3D structure of wild-type BcGH76 in complex with
MSMSMe 1, 1,6-ManDMJ 2, and 1,6-ManIFG 3 were solved
to resolutions of 1.30, 1.30, and 1.40 ꢀ, respectively, whilst the
structure of the BcGH76-D125N variant in complex with a-
,6-mannopentaose was solved at 1.20 ꢀ (Table S1). Consis-
tent with other released GH76 structures (including a lower-
resolution structure of WT BcGH76 (PDB ID: 4BOK ) and
a complex with a-1,6-mannobiose (4BOJ )) the native
1
2
3
[14]
with its role as a catalytic nucleophile. As expected, a D125N
variant was inactive toward a-1,6-Man due to the require-
[14]
3
structure of BcGH76 comprises an (a/a) helical barrel fold
ment for general acid catalysis.
6
with a long solvent-accessible cleft running laterally across the
face of the barrel (Figure 1). Complexes with a kinetically
identical engineered crystal-packing BcGH76 variant (see SI)
allowed identification of the active site and ꢀ4 to + 1 sugar-
The conformational distortions observed in both the
Michaelis and BcGH76-3 complexes are indicative of
O
°
1
a
S $B $ S conformational pathway for BcGH76 cat-
2
2,5
5
alysis. Ab initio QM/MM metadynamics simulations were
2
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used to quantify how BcGH76 alters the preferences of both
a-mannose and IFG for these higher energy conformers. The
calculations show that the enzyme dramatically reshapes the
energetically accessible conformational landscape of both the
[
11b]
[10e]
mannose
(
and IFG
moieties within the ꢀ1 subsite
4
Figure 2). Of special note, the undistorted C conformers are
1
no longer the global energy minima, and are situated at much
higher energies than the most stable distorted conformation
ꢀ
1
N
(
6 kcalmol higher compared to the S5/B2,5 conformer of
ꢀ1
O
IFG and > 15 kcalmol higher compared to the S2/B2,5
conformer of the a-mannoside at the ꢀ1 subsite in the
Michaelis complex). In sum, these data are consistent with
O
°
1
GH76 enzymes acting through a canonical S2$B2,5 $ S5
itinerary (Figure 3).
An unexpected interaction, apparently unique to
BcGH76, is observed within the Michaelis and BcGH76-3
complexes. In both structures the catalytic nucleophile
(
Asp124) makes a direct hydrogen bonding interaction with
the distal 3-OH group of the substrate and inhibitor (O–O
distance approx. 2.7 ꢀ, see Figures 1 and S4). These inter-
actions are also predicted for the lowest-energy conforma-
tions of the on-enzyme FELs for a-mannose and IFG
(
Figure 2). Hydrogen-bonding interactions with the ꢀ1
sugar 2-hydroxy and the nucleophile of retaining b-glucosi-
dases/b-xylanases have been shown to be important for
catalysis, contributing up to 10 kcalmol to TS stabiliza-
ꢀ
1
°
[
18]
tion. In the case of a-mannosidases with the ꢀ1 sugar in an
O
S conformation in the Michaelis complex, the 3-hydroxy
2
group is geometrically positioned to provide a similar inter-
action, and we speculate that this hydrogen bonding inter-
O
action facilitates deformation to a reactive S conformation
2
in the Michaelis complex, and is geometrically and electroni-
°
cally optimized to stabilize the B2,5 TS . Notably, such an
interaction is not possible for retaining b-mannosidases as the
catalytic nucleophile approaches the anomeric position from
the opposing (bottom) face of the substrate. Furthermore,
such an interaction is not possible for the metal-dependent a-
mannosidases of families GH38, 47, and 92, as the essential
2
+
2+
divalent metal (Ca or Zn ) bridges O2 and O3, which has
been speculated to assist distortion of the substrate into the
preactivated conformation required for catalysis, assist in
[
10d]
conformational transitions along the reaction coordinate,
°
[19]
and stabilize charge development on O2 at the TS .
Interestingly, an analogous interaction is present within
family GH38 enzymes in which the catalytic nucleophile
2
+ [19]
makes a direct interaction with Zn .
Intriguingly, this
interaction has remarkable precedent in a chemical glyco-
sylation involving the nucleophilic substitution of a 4,6-
benzylidene-protected a-mannosyl triflate; density functional
°
theory calculations of the TS based on kinetic isotope effects
implicated a B2,5 conformation characterized by the presence
Figure 2. QM/MM metadynamics simulation of ligand binding to
BcGH76 a-1,6-mannanase. A) Conformational free-energy landscape
FEL) of the mannoside moiety at the ꢀ1 enzyme subsite within the a-
,6-mannopentaose complex. B) Complex with azasugar 3 with the
of a hydrogen bond between the acceptor alcohol and O3 of
(
1
[20]
the donor.
Previous work has suggested that IFG-type inhibitors,
lowest free energy. C) Conformational free-energy landscape (FEL) of
4
which thus far have only been observed in C conformations
the isofagomine moiety at the ꢀ1 enzyme subsite. FELs contoured at
1
ꢀ1
1
kcalmol . Star denotes the coordinates of the conformation from
when bound to mannosidases (analyzed in Ref. [10e]), are
°
(B).
likely poor TS mimics due to the significant energy barrier
associated with interconversion to the B2,5 conformation (a
calculated FEL of IFG in solution shows B2,5 to be approx-
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.
Angewandte
Communications
Cal AutoiTC200 calorimeter (Malvern Instruments). 1,6-ManIFG
450 mm) was titrated into the ITC cell containing 36 mm BcGH76,
and the Kd value calculated using the Origin 7 software package
MicroCal). QM/MM MD simulations were performed using
(
(
a method that combines the Car-Parrinello MD, based on DFT,
with force-field MD. The IFG moiety of the 1,6-ManIFG inhibitor
and the a-mannoside at the ꢀ1 enzyme subsite were treated
quantum-mechanically (QM region), whereas the rest of the sub-
strate, the protein and the solvent (MM region) were treated with the
AMBER force field. The PBE functional was used for the DFT
calculations in view of its good performance on isolated sugars and
carbohydrate-active enzymes. The metadynamics algorithm was used
to explore the conformational free-energy landscapes of the IFG
moiety of protonated 3 and the a-mannoside ring at the ꢀ1 enzyme
subsite, taking as collective variables two of the puckering coordi-
nates of Cremer and Pople (q, f) (see SI for further details and
complete references).
Received: October 29, 2014
Revised: January 29, 2015
Published online: && &&, &&&&
Keywords: carbohydrates · computational chemistry ·
conformational analysis · enzymatic mechanisms ·
glycosidase inhibitors
.
Figure 3. Proposed reaction coordinate for GH76 retaining a-manna-
nases via a B2,5 TS . The deformed ground-state skew and transition-
state boat conformations are stabilized through a direct interaction
with the putative catalytic nucleophile (Asp124 for BcGH76).
°
[
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Experimental Section
Crystals of BcGH76 were grown as described in the Supporting
Information (SI), with 3D structures determined using X-ray
diffraction data collected at beamlines I04 and I04-1 of the Diamond
Light Source (Didcot, UK). Further details regarding data processing,
structure solution, and refinement are available from PDB headers
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and a-1,6-M , as described in the SI. Thermodynamic studies of 1,6-
3
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4
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Angewandte
Chemie
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

.
Angewandte
Communications
Communications
Conformational Analysis
Family GH76 endo-a-mannanases partic-
ipate in construction and breakdown of
fungal cell wall mannoprotein. A com-
bined synthetic, structural, and theoret-
ical study discloses the first inhibitors of
this family of enzymes and quantifies how
the enzyme distorts an azasugar inhibitor
into a transition-state-mimicking boat
conformation.
A. J. Thompson, G. Speciale,
J. Iglesias-Fernꢁndez, Z. Hakki, T. Belz,
A. Cartmell, R. J. Spears, E. Chandler,
M. J. Temple, J. Stepper, H. J. Gilbert,
C. Rovira,* S. J. Williams,*
G. J. Davies*
&&&& — &&&&
Evidence for a Boat Conformation at the
Transition State of GH76 a-1,6-
Mannanases—Key Enzymes in Bacterial
and Fungal Mannoprotein Metabolism
6
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
These are not the final page numbers!