An Esterase-like Lyase Catalyzes Acetate Elimination in Spirotetronate/Spirotetramate Biosynthesis

Article abstract of DOI:10.1002/anie.201812105

Spirotetronate and spirotetramate natural products include a multitude of compounds with potent antimicrobial and antitumor activities. Their biosynthesis incorporates many unusual biocatalytic steps, including regio- and stereo-specific modifications, cyclizations promoted by Diels–Alderases, and acetylation-elimination reactions. Here we focus on the acetate elimination catalyzed by AbyA5, implicated in the formation of the key Diels–Alder substrate to give the spirocyclic system of the antibiotic abyssomicin C. Using synthetic substrate analogues, it is shown that AbyA5 catalyzes stereospecific acetate elimination, establishing the (R)-tetronate acetate as a biosynthetic intermediate. The X-ray crystal structure of AbyA5, the first of an acetate-eliminating enzyme, reveals a deviant acetyl esterase fold. Molecular dynamics simulations and enzyme assays show the use of a His-Ser dyad to catalyze either elimination or hydrolysis, via disparate mechanisms, under substrate control.

Full text of DOI:10.1002/anie.201812105

Angewandte
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International Edition: DOI: 10.1002/anie.201812105
German Edition: DOI: 10.1002/ange.201812105
Biocatalysis
Hot Paper
An Esterase-like Lyase Catalyzes Acetate Elimination in
Spirotetronate/Spirotetramate Biosynthesis
Nicholas R. Lees+, Li-Chen Han+, Matthew J. Byrne+, Jonathan A. Davies, Alice E. Parnell,
Pollyanna E. J. Moreland, James E. M. Stach, Marc W. van der Kamp, Christine L. Willis,* and
Paul R. Race*
Abstract: Spirotetronate and spirotetramate natural products
include a multitude of compounds with potent antimicrobial
and antitumor activities. Their biosynthesis incorporates many
unusual biocatalytic steps, including regio- and stereo-specific
modifications, cyclizations promoted by Diels–Alderases, and
acetylation-elimination reactions. Here we focus on the acetate
elimination catalyzed by AbyA5, implicated in the formation
of the key Diels–Alder substrate to give the spirocyclic system
of the antibiotic abyssomicin C. Using synthetic substrate
analogues, it is shown that AbyA5 catalyzes stereospecific
acetate elimination, establishing the (R)-tetronate acetate as
a biosynthetic intermediate. The X-ray crystal structure of
AbyA5, the first of an acetate-eliminating enzyme, reveals
a deviant acetyl esterase fold. Molecular dynamics simulations
and enzyme assays show the use of a His-Ser dyad to catalyze
either elimination or hydrolysis, via disparate mechanisms,
under substrate control.
facilitated by the coordinated action of acetylases and
deacetylases, which function within stringently regulated
cellular networks.[2] Deacetylases have been the subject of
considerable detailed investigation, and have been shown,
without exception, to catalyze the hydrolysis of the acetate via
attack on the carbonyl group.[3] Recently, studies of spirotetr-
onate and spirotetramate biosynthetic pathways have
revealed an alternative biocatalytic route to the processing
of acetylated molecules. This involves the action of free-
standing acetate lyases; enzymes which employ a hitherto
uncharacterized mechanism to eliminate acetate with the
formation of a double bond (Scheme 1).[4] These enzymes are
Scheme 1. Enzyme-catalyzed deacetylation and acetate elimination.
A
cetylation is a ubiquitous chemical modification of major
importance in biology. Acetylation state impacts protein
stability, folding and localization, central metabolism, apop-
tosis, transcription, cytoskeletal organization, circadian regu-
lation, bacterial cell wall architecture and integrity, natural
product bioactivity, and antimicrobial resistance, amongst
others.[1] The addition or removal of acetyl groups is
unique to the spirotetronate/spirotetramate pathways, sharing
less than 20% sequence identity to any protein of known
structure. Given the role of acetate lyases in the biosynthesis
of natural products of outstanding clinical potential, their
enigmatic enzymology, and their potential utility as industri-
ally relevant biocatalysts, these enzymes represent intriguing
and important targets for detailed study.
[*] N. R. Lees,[+] Dr. L.-C. Han,[+] J. A. Davies, Prof. C. L. Willis
School of Chemistry, University of Bristol
Bristol, BS8 1TS (UK)
To elucidate the mechanistic details of enzyme catalyzed
acetate elimination, we focused on the putative acetate lyase
AbyA5 from the abyssomicin C biosynthetic pathway. The
antimicrobial natural product abyssomicin C (1) is a potent
inhibitor of bacterial folate metabolism and is effective
against a multitude of Gram-positive pathogens, including
Mycobacterium tuberculosis and multi-drug resistant strains
of Staphylococcus aureus.[5] The biosynthetic pathway to this
compound, which comprises a multi-modular polyketide
synthase (PKS) and associated tailoring and regulatory
proteins, is encoded for within a single gene cluster (aby)
spanning approximately 60 kb of the genome of the marine
actinomycete Verrucosispora maris AB-18-032.[6] Based on
feeding studies, chemotyping of V. maris gene knock-out
mutants, comparative bioinformatics analyses, and in vitro
studies of homologous enzymes from other spirotetronate
and spirotetramate pathways, a general mechanism for the
biosynthesis of abyssomicin C has been proposed (Supporting
Information, Figure S1).[7] One of the most intriguing features
of this pathway is the formation and subsequent tailoring of
the tetronate-ring-containing compound 2, via the acetylated
intermediate 3, to yield 4, which subsequently serves as
E-mail: Chris.Willis@bristol.ac.uk
Dr. M. J. Byrne,[+] Dr. A. E. Parnell, Dr. M. W. van der Kamp,
Dr. P. R. Race
School of Biochemistry, University of Bristol
Bristol, BS8 1TD (UK)
E-mail: Paul.Race@bristol.ac.uk
N. R. Lees,[+] Dr. L.-C. Han,[+] Dr. M. J. Byrne,[+] J. A. Davies,
Dr. A. E. Parnell, Dr. M. W. van der Kamp, Prof. C. L. Willis,
Dr. P. R. Race
BrisSynBio Synthetic Biology Research Centre, University of Bristol
Bristol, BS8 1TQ (UK)
P. E. J. Moreland, Dr. J. E. M. Stach
School of Biology, Newcastle University
Newcastle-upon-Tyne, NE1 7RU (UK),
and
Centre for Synthetic Biology and the Bioeconomy
Newcastle-upon-Tyne, NE2 4AX (UK)
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201812105.
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a substrate for a Diels–Alderase-catalyzed intramolecular
[4+2] cycloaddition reaction (Figure 1).[8] The conversion of 3
to 4 is postulated to proceed via the elimination of acetate,
yielding the 14,15-exocyclic double bond, but it is not known
which enantiomer of 3 is involved. The introduction of this
dienophile is implicitly required for the subsequent AbyU-
catalyzed cycloaddition reaction that forms the heterobicyclic
ring nucleus of abyssomicin C.
whether AbyA5 does indeed catalyze acetate elimination,
this enzyme was recombinantly over-expressed in E. coli
BL21(DE3) cells and purified to homogeneity (Supporting
Information, Figure S3). Recombinant AbyA5 was found to
be a monomeric, well-folded, monodisperse species in
solution.
Both enantiomers of the unnatural substrate analogues of
acetate 3 were synthesized. Hydroxy ester 5 was readily
prepared from d-mannitol diacetonide in 4 steps according to
the literature.[9] Coupling with b-ketothioester 6 in the
presence of CF3CO2Ag gave keto ester 7 in 79% yield. The
key TBAF-mediated Dieckmann cyclization has wide prece-
dent in the literature, however, difficulty has been reported
when using this method.[10] In our hands this reaction proved
challenging until the purified compound was washed with 1m
HCl according to Osada et al.[11] Following the cyclization, the
primary alcohol 8 was acetylated to give the desired (R)-
analogue (R)-9. To prepare a standard of the elimination
product 10 for the enzyme assays, acetate (R)-9 was reacted
with DBU giving alkene 10, other bases (for example, TBAF,
imidazole, and triethylamine) gave no reaction. An alterna-
tive approach to the synthesis of 10 is from 11 as shown in
Scheme 2.
The incubation of (R)-acetate (R)-9 with recombinant
AbyA5 in vitro yielded a single product with a mass (m/z
[MÀH]À = 237) and 1H-NMR in keeping with alkene 10, and
consistent with the authentic synthetic standard (Figure 2 and
Supporting Information, Figure S4), confirming that elimina-
tion had taken place. Control reactions lacking enzyme, or
containing heat denatured AbyA5, showed no evidence of
product formation. AbyA5 showed no activity against the (S)-
enantiomer (S)-9, demonstrating that the enzyme is stereo-
specific for the (R)-form only. Steady-state kinetic character-
ization of AbyA5 with (R)-9, employing a spectrophotometric
assay (See the Supporting Information for details), yielded
Figure 1. Tetronate ring formation and tailoring during abyssomicin C
biosynthesis.
Amino acid sequence alignments of the known spirotetr-
onate/spirotetramate acetate lyases Agg5 (agglomerin)[4b] and
QmnD4 (quartromicin),[4a] and putative acetate-eliminating
enzymes from related biosynthetic pathways, with open
reading frames within the aby cluster, identify AbyA5 as
the likely eliminating enzyme from the abyssomicin C path-
way (Supporting Information, Figure S2). To ascertain
k
cat = 1.8 Æ 0.13 minÀ1, Km = 27 Æ 8.4 mm, and kcat/Km = 0.072 Æ
0.021 minÀ1 mm (Figure 2). Together these data demonstrate
Scheme 2. Synthesis of acetate-elimination precursor (R)-9 and product 10. MS=molecular sieve, THF=tetrahydrofuran, TBAF=tetrabutylammo-
nium fluoride, DMAP=4-(dimethylamino)pyridine, DBU=1,8-diazabicyclo[5.4.0]undec-7-ene, LDA=lithium diisopropylamide, DMP=Dess–
Martin periodinane, DMSO=dimethylsulfoxide.
2
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wavelength anomalous dispersion (SAD) method, to 2.3 ꢀ
resolution, employing a selenomethinone-labeled quadruple
point mutant of AbyA5, within which the residues Leu66,
Leu158, Leu195, and Leu295 were mutated to methionines
(See the Supporting Information for details). This structure
was subsequently used as a molecular-replacement search
model to determine that of wild type AbyA5 to 2.5 ꢀ
resolution. The structure of AbyA5 is consistent with
a monomeric a/b hydrolase (ABH), possessing a central,
largely parallel eight-stranded b-sheet surrounded by a-
helices (Figure 3). As with other ABHs, the second strand
Figure 3. X-ray crystal structure of AbyA5. a) Overall fold of the AbyA5
monomer showing the catalytic domain (blue) and four-helix capping
domain (yellow). S, Ser198; D, Asp 285; H, H312; b) Active site cleft
on the enzyme surface (red). The capping domain has been removed
for clarity. Asp285 is obscured by His312. c) Model of (R)-3 and (S)-3
docked into the active site of AbyA5.
within the b-sheet runs antiparallel to the remaining seven
and the sheet possesses a left-handed super-helical twist. The
major structural features that distinguish AbyA5 from other
ABHs are the large number of helices that decorate the
central b-sheet core of the enzyme (15 in total) and the
presence of a four-helix subdomain that extends outwards
from the top of AbyA5 in a fashion analogous to that reported
in 2,6-dihydroxy-pseudo-oxynicotine hydrolase.[12] Structur-
ally, AbyA5 is most closely related to members of the acetyl
esterase family of deacetylases, despite minimal amino acid
sequence identity (less than 20%). Inspection of the AbyA5
crystal structure and superposition with structurally related
acetyl esterases unambiguously identifies the location of the
enzyme active site, which sits within an extended cleft on the
surface of the enzyme of approximately 40 ꢀ in length and
approximately 10 ꢀ in depth. The left side of the cleft houses
a canonical ABH catalytic triad, comprising of the residues
Ser198, Asp285, and His312 (Figure 3). Ser198 is located on
a nucleophilic elbow formed by a loop linking b5 and a7.
His312 sits on the opposite side of Ser198, on a loop between
b8 and a14, directly above Asp285.
Figure 2. Acetate-eliminating activity of AbyA5. a) Synthesized sub-
strate analogues (R)-9, (S)-9, and alkene 10. b) HPLC-MS demonstrat-
ing the AbyA5-catalyzed conversion of (R)-9 to 10. c) Steady-state
kinetic characterization of the conversion of (R)-9 to 10. Error bars are
standard errors from the mean calculated from reactions run in
triplicate.
the eliminating activity of AbyA5, unambiguously establish
the role of this enzyme in abyssomicin C biosynthesis, and
reveal that this reaction proceeds in a stereospecific manner.
To provide a structural framework of the AbyA5-cata-
lyzed elimination reaction, the X-ray crystal structure of the
enzyme was determined. This was achieved using the single-
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To further investigate the acetyl elimination by AbyA5,
a modelling study was performed. Molecular docking was
conducted using the AbyA5 crystal structure with both (R)-3
and (S)-3. This yielded a series of closely related models of the
AbyA5–substrate complex, each of which positions the
substrate directly within the AbyA5 active site cleft and
locates the acetyl group in close proximity to Ser198 and
His312 (Figure 3). The side chain of 3 occupies a largely
hydrophobic portion on the right of the active site cleft. Given
the minimal number of contacts between AbyA5 and the
substrate in this region, it appears unlikely that chain length
and composition is a major determinant of substrate selec-
tivity. This is consistent with our in vitro assay data, which
demonstrate that the substrate analogue (R)-9, which lacks
the side chain functional group decoration of 3, is readily
acted upon by the enzyme. In contrast, appropriate position-
ing of the acetylated tetronate ring within the active site
appears to play a significant role in substrate binding. This is
achieved by a combination of shape and charge complemen-
tarity between the acetylated ring and the active site cavity,
supported by several hydrogen bonds. The point mutants
AbyA5_H321A and Ser198A showed no activity with (R)-9,
despite both proteins being well-folded monodisperse species
in solution (Supporting Information, Figure S5), implying
a critical role for these residues in substrate positioning and/or
catalysis within the active site. Molecular dynamics simula-
tions using docked poses of (R)-3 reveal a distance of
approximately 3.5 ꢀ between His312 and C15 of the sub-
strate, optimal for proton abstraction and consistent with the
His-dependent catalytic mechanism proposed previously for
QmnD4 (Supporting Information, Figure S6).[4a] The distance
between His312 and C15 for (S)-3 docked poses is greater
than 4.5 ꢀ, sufficient to preclude proton abstraction and
thereby negate catalysis (Figure 3). Together, these data offer
an explanation for the stereoselectivity of AbyA5 observed in
our in vitro enzyme assays.
The crystal structure of AbyA5 raises the intriguing
possibility that this protein scaffold could support both
acetate elimination and hydrolysis, though no (R)-9 hydro-
lytic product 8 was detected in our functional assays. To test
this hypothesis, the ability of AbyA5 to deacetylate p-
nitrophenolacetate (p-NPA) in vitro was monitored spectro-
photometrically.[13] AbyA5 was found to catalyze the hydro-
lytic deacetylation of p-NPA, with kcat = 44 Æ 2.4 minÀ1, Km =
78 Æ 9.2 mm, and kcat/Km = 0.62 Æ 0.31 minÀ1 mm (Supporting
Information, Figure S7). Molecular dynamics simulations of
AbyA5, in the absence of substrate, reveal the appropriate
positioning of the His-Ser-Asp triad, as defined by the
occupation of hydrogen bonds between these side chains,
for approximately 10% of the simulation duration, consistent
with the ability of the enzyme to catalyze ester bond cleavage
via a canonical acetyl esterase mechanism (Supporting
Information, Figure S8).[14] The infrequent adoption of this
catalytically competent state accounts for the comparatively
poor catalytic efficiency of AbyA5 for p-NPA as compared to
naturally evolved acetyl esterases, with an up to 60-fold lower
kcat/Km (Supporting Information, Table S2). Neither
AbyA5_H321A nor Ser198A showed any activity with p-
NPA. Docking studies confirmed that p-NPA can be readily
accommodated within the AbyA5 active site.
In summary, we report the structural and functional
characterization of the acetate lyase AbyA5, revealing the
molecular details of the acetate-elimination reaction cata-
lyzed by this enzyme, and in doing so, establish explicitly the
role of this enzyme in abyssomicin C biosynthesis. AbyA5 is
shown to possess an acetyl-esterase-like fold, within which
conserved catalytic machinery can be deployed to facilitate
either acetate elimination or ester hydrolysis, in a manner
dictated by substrate identity. Our studies establish the origins
of substrate selectivity in AbyA5, revealing absolute stereo-
selectivity for the (R)-tetronate, but relaxed selectivity for the
C3 chain. These findings hint at the potential general utility of
AbyA5 as an acetate-elimination biocatalyst. Although
evolutionarily selected to catalyze elimination, AbyA5 exhib-
its kinetic parameters for ester hydrolysis comparable to
naturally evolved acetyl esterases. Catalytic multifunctional-
ity is an inherent feature of many biocatalysts; however,
AbyA5 is unusual in its proficiency in performing a secondary
non-cognate reaction.[15] These studies also further expand the
breadth of transformations catalyzed by a/b-hydrolase fold
enzymes, highlighting the utility of this protein scaffold in
supporting a diverse array of biocatalytic reactions. Finally,
given the high degree of sequence identity between AbyA5
and acetate-eliminating enzymes from other spirotetronate/
spirotetramate pathways, we conclude that many of the key
findings reported herein will be directly applicable to acetate
lyases from other biosynthetic pathways.
Acknowledgements
This study was supported by BBSRC and EPSRC through the
BrisSynBio Synthetic Biology Research Centre (BB/
L01386X/1), PhD studentships awarded to N.R.L. (EPSRC,
EP/G036764/1 and GSK), M.J.B. (BBSRC, BB/D526037/1),
J.A.D. (EPSRC, EP/L015366/1), P.E.J.M. (BBSRC, BB/
J014516/1), and a BBSRC David Phillips Fellowship to
M.W.v.d.K (BB/M026280/1).
Conflict of interest
The authors declare no conflict of interest.
Keywords: antibiotics · biocatalysis · enzyme structure ·
enzymology · polyketides
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Manuscript received: October 24, 2018
Version of record online: && &&, &&&&
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Communications
Biocatalysis
To eliminate, or to hydrolyze, that is the
question: Structural, mechanistic, and
computational studies of the abyssomi-
cin C pathway enzyme AbyA5 establish
the molecular origins of enzyme-cata-
lyzed acetate elimination. The unexpected
acetyl-esterase-like scaffold of the protein
is shown to support both acetate elimi-
nation and ester hydrolysis, in a manner
dictated by substrate identity.
N. R. Lees, L.-C. Han, M. J. Byrne,
J. A. Davies, A. E. Parnell,
P. E. J. Moreland, J. E. M. Stach,
M. W. van der Kamp, C. L. Willis,*
P. R. Race*
&&& — &&&
An Esterase-like Lyase Catalyzes Acetate
Elimination in Spirotetronate/
Spirotetramate Biosynthesis
6
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