Structural and mechanistic insights into a Bacteroides vulgatus retaining N-acetyl-β-galactosaminidase that uses neighbouring group participation

Article abstract of DOI:10.1039/c6cc04649e

Bacteroides vulgatus is a member of the human microbiota whose abundance is increased in patients with Crohn's disease. We show that a B. vulgatus glycoside hydrolase from the carbohydrate active enzyme family GH123, BvGH123, is an N-acetyl-β-galactosamin

Full text of DOI:10.1039/c6cc04649e

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Structural and mechanistic insights into a Bacteroides vulgatus
retaining N-acetyl-β-galactosaminidase that uses neighboring
group participation
Received 00th January 20xx,
Accepted 00th January 20xx
a
b
c,d
c,d,*
a,*
b,*
DOI: 10.1039/x0xx00000x
C. Roth, M. Petricevic, A. John, E.D. Goddard-Borger,
G.J. Davies and S. J. Williams
www.rsc.org/
not found.Error! Reference source not found.Error! Reference source not found.
Bacteroides vulgatus is a member of the human microbiota
In
whose abundance is increased in patients with Crohn's addition, B. vulgatus and other members of the Bacteroidetes
disease. We show that a B. vulgatus glycoside hydrolase from have specialised loci for the degradation of intestinal mucosal
5
the carbohydrate active enzyme family GH123, BvGH123, is glycoproteins and the glycocalyx. However, it appears that the
an N-acetyl-β-galactosaminidase that acts with retention of ability of Bacteroides spp. to degrade host glycoconjugates is
stereochemistry, and through a 3-D structure in complex with also enabled by enzymes encoded outside of the PULs. B.
Gal-thiazoline, provide evidence in support of a neighbouring vulgatus encodes a single copy of an enzyme from glycoside
Error! Reference source not found.
group participation mechanism.
hydrolase family GH123;
a poorly
characterized family with an emerging ability to degrade hostꢀ
The human gastrointestinal tract undergoes a constant process derived glycoconjugates. In this work we demonstrate that
7
of renewal, leading to production of 5 × 10 cells per minute previously uncharacterized protein BvGH123 is a
Nꢀacetylꢀβꢀ
1
and the discharge of 250 g of cells per day. Colonocytes galactosaminidase. We experimentally demonstrate for the first
derived from the epithelia of the large intestine can be isolated time that a member of this family hydrolyses glycosides with
at concentrations of up to 10 per gram of wet fecal material,
7
2
retention of anomeric stereochemistry, and provide evidence for
and are an important nutrient source for the gut microbiota. A a neighbouring group participation mechanism through an Xꢀ
wide range of carbohydrates are represented on intestinal ray crystal structure in complex with an inhibitor that mimics
epithelial cells, including polysaccharides, glycoproteins and the proposed intermediate of the reaction mechanism.
glycolipids. B. vulgatus is a widespread member of the human
microbiota, and is generally considered beneficial, although it is Paenibacillus sp., and CpNga123 from C. perfringens (whose
an occasional opportunistic pathogen. B. vulgatus is typically structure was recently solved) have led to the proposal of a
Previous studies of GH123 members, initially NagP from
6
7
present at levels of around 4% of bacteria in the gut microbiota, twoꢀstep neighbouring group participation mechanism for
but can rise to constitute >40% of the gut microflora in Crohn's catalysis (Figure 1). According to such a mechanism, the
3
disease patients. B. vulgatus, like other Bacteroides spp., enzyme possesses two key active site residues acting in the role
8
possesses specialized polysaccharide utilization loci (PULs) of acid/base and transition state stabilization. In the first step,
encoding complex enzymatic machineries able to degrade and nucleophilic attack by the 2ꢀacetamido group oxygen on the
harness a wide variety of complex dietary polysaccharides anomeric centre, assisted by charge stabilization and orientation
along with SusC/SusD orthologues capable of importing by the transition state stabilizing residue, occurs simultaneously
4
Error! Reference source
oligosaccharide fragments into the periplasm.
with protonation of the glycosidic oxygen by the acid/base
residue to assist leaving group departure, resulting in the
formation of an oxazolinium ion. In the second step, the
acid/base residue acts as a base to assist in deprotonation of a
nucleophilic water molecule, while the stabilizing residue
9
facilitates ringꢀopening of the oxazolinium ion intermediate.
Evidence in support of this mechanism was initially based on
6
the nanomolar inhibition of NagP by Galꢀthiazoline; an
inhibitor that mimics the proposed oxazolinium ion
1
0
intermediate (more formally a closelyꢀrelated transitionꢀstate),
along with the proposal of a catalytic diad of adjacent carboxyl
residues reminiscent of those seen in βꢀhexosaminidases of
This journal is © The Royal Society of Chemistry 2016
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GH20 and GH84, which are known to act through a at
neighbouring group participation mechanism.
δ
5.22 ppm characterized by a small (
Recently, the constant. These experimental data are consistent with the initial
first Xꢀray structure of a GH123 enzyme, CpNga123, in a formation of βꢀGalNAc ( ), revealing that BvGH123, and by
J = 4 Hz) coupling
1
1,12
2
β
product complex and a pseudoꢀMichaelis complex of an inference all GH123 members, act with a net retention of
inactive mutant with substrate confirmed the identity of the anomeric configuration. Using isothermal titration calorimetry,
1
4
catalytic diad, and revealed binding orientations of the 2ꢀ Galꢀthiazoline was shown to bind BvGH123 with
D
K = 9.6
acetamido group that while not oriented directly below C1, was nM (Figure S2b). Collectively, these results support previous
positioned in a manner consistent with that needed for suggestions that enzymes of family GH123 use a neighbouring
7
neighbouring group participation.
group participation mechanism involving the 2ꢀacetamido
group.
acid/base
O
H
O
O
O
H
-
+
O
δ
δ
O
O
R
OR
O
-
HN
+
HN
O δ
2
H O
δ
ROH
H
3
C
O
O
O
O
CH3
O
O
stabilizing residue
H
O
O
H
HN
O
O
O
3
H C
O
H
O
O
O
H
oxazolinium
intermediate
-
+
O
δ
δ
O
O
H
OH
HN
O
+
O δ-
CH3
δ
HN
CH
3
O
O
O
O
Fig. 1 Neighbouring group participation mechanism by a retaining N-acetyl-
β-hexosaminidase. Hydroxyl groups have been omitted for clarity.
1
Fig. 2 Stacked H NMR spectra showing time course of BvGH123 hydrolysis
of PNPGalNAc. Substrate hydrolysis results in initial release of β-GalNAc with
To further investigate the mechanism of the GH123 retention of anomeric configuration, which then undergoes mutarotation.
galactosaminidases, residues 20ꢀ582 of ABR39589.1, encoding
Table 1. 3-D structure data for BvGH123.
BVU_2198, without its signal peptide (hereafter BvGH123)
BvGH123–
BvGH123–Gal-
thiazoline
was expressed in E. coli (Figure S1). Size exclusion
chromatography–multiꢀangle laser light scattering revealed
BvGH123 to be a dimer in solution. The enzyme displays a pH
Native (apo)
P2
GlcNAc
Space group
1
2
1
2
1
P2
1
2
1
2
1
P2
1 1 1
2 2
Resolution limit
1.85 Å
0.141(1.930)
9.9(1.2)
2.1 Å
2.3 Å
optimum
of
5
using
4ꢀnitrophenyl
NꢀacetylꢀβꢀDꢀ
R
merge
0.104(1.530)
12.0(1.2)
100(100)
0.014
0.063(1.552)
15.6(1.1)
93.4(63.6)
0.013
galactosaminide (PNPGalNAc,
1) as a substrate (Figure S2a).
I/σI
The kinetic preference for βꢀgalactosaminides versus βꢀ
glucosaminides was determined using PNPGalNAc and 4ꢀ
Completeness (%)
R.m.s.d bond
lengths (Å)
R.m.s.d bond
angles (º)
99.9(100)
0.016
nitrophenyl
PNPGalNAc,
PNPGlcNAc,
N
ꢀacetylꢀβꢀ
Dꢀglucosaminide (PNPGlcNAc). For
3
–
1
–1
k
cat
/
K
M
= 8.0±0.4 × 10
M
s
and for
1.70
1.59
1.63
2
–
1
–1
k
cat
/
K
M
= 3.8±0.6 × 10 M s ; saturation could
not be achieved, preventing the accurate determination of
and values. These data indicate a 21ꢀfold preference for βꢀ
galactosaminides, consistent with previous reports on NagP
k
cat
K
M
6
At the time of our structure solution, no GH123 structure
had been reported and thus the threeꢀdimensional structure of
BvGH123 was solved by experimental phasing using single
anomalous diffraction from a selenomethionineꢀsubstituted
7
and CpNga123, providing further evidence that the enzymes of
this family are dedicated ꢀacetylꢀβꢀgalactosaminidases.
N
Previous studies have only speculated on the stereochemical derivative and subsequently refined against a native dataset to a
outcome of substrate hydrolysis, which is of obvious resolution of 1.85 Å (Table 1, S1). The overall structure reveals
importance for proposal of
mechanism.
a
feasible enzymatic a two domain structure with an Nꢀterminal βꢀsheet domain
1
3
Error! Reference source not found.
1
We used H NMR (residues 1ꢀ195) followed by a catalytic (β/α) ꢀbarrel domain
8
spectroscopy to monitor the stereochemical outcome of the (residues 196ꢀ563) (Fig 3a). The latter has three insertions
enzymeꢀcatalyzed hydrolysis of PNPGalNAc (
Upon addition of enzyme, rapid consumption of substrate led to cap, as observed recently in the CpGH123 structure. Structures
1
) (Figure 2). forming a 4ꢀstranded βꢀwing domain and a Cꢀterminal αꢀhelical
7
the production of a new product, with an anomeric signal at
δ
of BvGH123 and CpNGA123 overlap with an r.m.s.d. of
4
.63 ppm that displayed a large ( = 8 Hz) coupling constant. approximately 1.7 Å over 500 residues. Differences include a
J
Over time, mutarotation led to the formation of a mixture of the longer Nꢀterminus for BvGH123, and a Cꢀterminal βꢀstrand
first compound, and a new compound with an anomeric signal insertion followed by a longer Cꢀterminal helix for CpNg123.
2
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Additionally a disulfide bond joining Cys117 and Cys430 is upon engagement with carbohydrate. In the case of BvGH123
observed only in BvGH123, connecting the loop comprising upon binding to Galꢀthiazoline the loop containing D361 and
residues 110ꢀ120 with the 430ꢀloop which is part of the active E362 moves to engage these acidic side chains into a closed
site. Sequence conservation within the GH123 family is conformation in which the catalytic residues engage in close
relatively low and is mainly confined to the catalytic residues contacts with the inhibitor. As the inhibitor is a mimic of the
Error!
and sections of the (β/α)
domain, found in both structures, shows weak structural
8
ꢀbarrel. The Nꢀterminal βꢀsheet oxazolinium intermediate (or possibly the transition state),
Reference source not found.
these interactions appear to represent those
similarity to the IgGꢀfold found in several antigens and of a catalytically competent form of the enzyme.
adhesion modules, as well as within Titin, and might facilitate
adhesion to the cell surface of the host, helping to degrade cell
1
5
surface glycans. Remote structural similarity is also observed
for the BACONꢀdomain found, for example, in GH5
Error! Reference source not found.
enzymes.
A stable assembly in the
1
6
crystal lattice identified by PISA is formed by interaction of
the 4 stranded βꢀwing domain formed by insertions between the
-
second and third strands and the helix of the (β/α) ꢀbarrel Fig. 4 BvGH123 undergoes conformational changes upon ligand binding.
8
Comparison of the conformations for the active site residues in: a) fully open
(
Figure S3), presumably representing the dimer observed in
form of apo BvGH123; b) partially closed form in complex with GalNAc; c)
fully closed form in complex with Gal-thiazoline.
solution.
The Xꢀray structures of the inhibitor and product complexes
of BvGH123 provide an intimate view of the geometry of the
active site. In the BvGH123–Galꢀthiazoline complex, which is
in a fully closed state, the adjacent carboxyl residues D361 and
E362 adopt positions consistent with roles as acid/base and
transition state stabilizer (Figure 3c). The BvGH123–GalNAc
product complex revealed only a partially closed conformation
with W306 nestled above the pyranose and D361 engaging with
the acetamido group of GalNAc (Figure 3b). This complex
contrasts with that reported for GalNAc bound to CpNga123,
7
which is in an open conformation (Figure S4). The pyranose
4
ring of Galꢀthiazoline adopts a
C
1
conformation consistent
1
4
4
with an expected
S
3
→
H
3
→
C
1
1
conformational itinerary for
7
the glycosylation halfꢀreaction. Complexes of CpNga123ꢀ
D344N/E345Q with substrate (ganglioside glycans GA2 or
4
Gb4) were observed in
a
C
1
conformation, which is
inconsistent with the substrate distortion expected with a
pseudoꢀaxial orientation of the anomeric group, as anticipated
7
for stereoelectronic reasons. Combined with the location of the
Fig. 3 Structures of Bacteroides vulgatus GH123 and complexes with Gal-
thiazoline and GalNAc. (a) Overview of the BvGH123 structure in complex
with Gal-thiazoline, rainbow-coloured from the N- to C-terminus Gal-
thiazoline is shown as space filled van der Waals surface. Views of the active
site of BvGH123 in complex with (b) GalNAc and (c) Gal-thiazoline,
2
ꢀacetamido group away from C1, the Michaelis complexes
obtained with CpNga123 have been suggested to represent nonꢀ
catalyticallyꢀcompetent complexes in which the open
conformation prevents the active centre residues from fully
o c
highlighting the active site residues. The 2mF -DF
m3 ap for GalNAc in blue is engaging with the substrate.
3
contoured at 0.22 e/Å (1σ r.m.s.d.) and 0.25 e/Å (1.5σ r.m.s.d.) for Gal-
thiazoline. The C1-O interaction of the N-acetyl group in the GalNAc complex
is highlighted with a red dotted line. (d) Interaction plot of Gal-thiazoline
within the active site.
To gain insight into the structural basis of the retaining
mechanism of BvGH123, we solved structures of complexes
with the product GalNAc (Figure 3b), as well as Galꢀthiazoline
at resolutions of 2.1 and 2.3 Å, respectively (Figure 3c).
Compared to the “apo” form, both complexes revealed a
movement both of several of the loops lining the active site and
of key residues, which is most pronounced for W306 and the
adjacent pair of catalytic residues D361 and E362 (Figure 4,
Figure S4). Similar movements were observed for CpNga123
Fig. 5 Overlay of BvGH123–Gal-thiazoline active site residues (wheat) with
members of other neighbouring group participation enzyme families (GH18,
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0, 25, 84, 85). (a) Overlay of family representatives with adjacent catalytic
498; d) J. Larsbrink, A. J. Thompson, M. Lundqvist, J.
G. Gardner, G. J. Davies and H. Brumer, Mol. Microbiol.,
2014, 94, 418; e) G. R. Hemsworth, G. Dejean, G. J.
Davies and H. Brumer, Biochem. Soc. Trans., 2016, 44,
1
8
residues: GH20 Paenibacillus sp. β-HexNAcase (purple, PDB: 3SUR), and
GH84 Bacteroides thetaiotaomicron O-GlcNAcase (light green, PDB: 2CHN),
11
which contain adjacent. (b) Overlay of family representatives with non-
9
4.
adjacent catalytic residues: GH18 Serratia marcescens chitinase A (grey,
19
5
6
7
N. A. Pudlo, K. Urs, S. S. Kumar, J. B. German, D. A.
Mills and E. C. Martens, mBio, 2015, 6.
T. Sumida, K. Fujimoto and M. Ito, J. Biol. Chem., 2011,
PDB: 2WK2), GH25 Aspergillus fumigatus lysozyme (steel blue, PDB:
2
0
21
2
X8R), and GH85 Streptococcus pneumoniae endo-D (plum, PDB: 2W92).
2
86, 14065.
A comparison of the active sites of GH families that are
I. Noach, B. Pluvinage, C. Laurie, K. T. Abe, M. G.
Alteen, D. J. Vocadlo and A. B. Boraston, J. Mol. Biol.,
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2
known to utilise a substrateꢀassisted retaining mechanism,
which includes GH18, 20, 25, 84 and 85, reveal a remarkable
spatial conservation of the catalytic residues located on the
8
9
1
008, 12, 539.
fourth βꢀstrand of the respective (β/α) ꢀbarrel (Figure 4).
8
S. J. Williams, B. Mark, D. J. Vocadlo, M. N. James and
S. G. Withers, J. Biol. Chem., 2002, 277, 40055.
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Families GH20 and 84, like GH123, have the catalytic
acid/base and stabilizer adjacent in their protein chains, and
have essentially identical arrangements of these groups and the
linking peptide backbone. Families GH18, 25 and 85 have a
one residue insertion between the catalytic residues, yet still
adopt a remarkably similar spatial arrangement. Likewise the
active site closure motion now observed for two GH123
enzymes is also seen for a GH20 hexoaminidase and a GH84
0
1
1
1
1
2
3
7
,12,22
OꢀGlcNAcase.
These hexosaminidase families have little
or no sequence conservation yet display similar folds and
arrangement of catalytic residues and use a neighbouring group
participation mechanism, suggesting a common solution to the
2
3
chemical problem of hydrolysis of Nꢀacetylꢀβꢀhexosaminides.
This work was supported by the Australian Research
Council (FT130100103), and the BBSRC (BB/K003836/1), and
the Ramaciotti Foundation and VESKI with additional support
from the Australian Cancer Research Foundation and Victorian 14
S. Knapp and D. S. Myers, J. Org. Chem., 2002, 67, 2995.
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T. Sumida, K. A. Stubbs, M. Ito and S. Yokoyama, Org.
Biomol. Chem., 2012, 10, 2607.
1
5
State Government Operational Infrastructure Support, NHMRC
IRIISS grant 9000220. We thank the Diamond Light Source for
access to beamlines I02, I03 and I04 (proposal number mxꢀ
1
1
6
7
9948) that contributed to the results presented here. We also
thank Dr. Andrew Leech at the Bioscience Technology Facility 18
in the Department of Biology in York for his help with the
1
9
J. M. Macdonald, C. A. Tarling, E. J. Taylor, R. J. Dennis,
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SECꢀMALLS experiments. We are grateful to Dr. Johan
Turkenburg and Sam Hart for assistance during data collection
and Wendy Offen for help with the crystallisation.
2
0
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