Structure and activity of the streptomyces coelicolor A3(2) β-N-acetylhexosaminidase provides further insight into GH20 family catalysis and inhibition

Article abstract of DOI:10.1021/bi401697j

β-N-acetylhexosaminidases (HEX) are glycosidases that catalyze the glycosidic linkage hydrolysis of gluco- and galacto-configured N-acetyl-β-d-hexosaminides. These enzymes are important in human physiology and are candidates for the biocatalytic production of carbohydrates and glycomimetics. In this study, the three-dimensional structure of the wild-type and catalytically impaired E302Q HEX variant from the soil bacterium Streptomyces coelicolor A3(2) (ScHEX) were solved in ligand-free forms and in the presence of 6-acetamido-6-deoxy-castanospermine (6-Ac-Cas). The E302Q variant was also trapped as an intermediate with oxazoline bound to the active center. Crystallographic evidence highlights structural variations in the loop 3 environment, suggesting conformational heterogeneity for important active-site residues of this GH20 family member. The enzyme was investigated for its β-N-acetylhexosaminidase activity toward chitooligomers and pNP-acetyl gluco- and galacto-configured N-acetyl hexosaminides. Kinetic analyses confirm the β(1-4) glycosidic linkage substrate preference, and HPLC profiles support an exoglycosidase mechanism, where the enzyme cleaves sugars from the nonreducing end of substrates. ScHEX possesses significant activity toward chitooligosaccharides of varying degrees of polymerization, and the final hydrolytic reaction yielded pure GlcNAc without any byproduct, promising high applicability for the enzymatic production of this highly valued chemical. Thermostability and activation assays further suggest efficient conditions applicable to the enzymatic production of GlcNAc from chitooligomers.

Full text of DOI:10.1021/bi401697j

Article
pubs.acs.org/biochemistry
Structure and Activity of the Streptomyces coelicolor A3(2)
β‑N‑Acetylhexosaminidase Provides Further Insight into GH20 Family
Catalysis and Inhibition
Nhung Nguyen Thi,†
,‡,§,⊥,∥
Wendy A. Offen, Franco̧
is Shareck, Gideon J. Davies,○
○
†
,†,‡,§
and Nicolas Doucet*
†
INRS-Institut Armand-Frappier, Universite
́
du Queb
ec Network for Research on Protein Function, Structure, and Engineering, 1045 Avenue de la Med
Laval, Quebec, Quebec G1V 0A6, Canada
GRASP, the Groupe de Recherche Axe sur la Structure des Protei
Montreal, Quebec H3G 0B1, Canada
Military Institute of Science and Technology, 17 Hoang Sam, Hanoi, Vietnam
́ ́
ec, 531 Boul. des Prairies, Laval, Quebec H7V 1B7, Canada
‡
PROTEO, the Queb
Universite
́
́
ecine,
́
́
́
§
́
́
nes, 3649 Promenade Sir William Osler, McGill University,
́
́
⊥
∥
Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Hanoi, Vietnam
○
Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, United Kingdom
S Supporting Information
*
ABSTRACT: β-N-acetylhexosaminidases (HEX) are glycosidases that catalyze the glycosidic linkage hydrolysis of gluco- and
galacto-configured N-acetyl-β-D-hexosaminides. These enzymes are important in human physiology and are candidates for the
biocatalytic production of carbohydrates and glycomimetics. In this study, the three-dimensional structure of the wild-type and
catalytically impaired E302Q HEX variant from the soil bacterium Streptomyces coelicolor A3(2) (ScHEX) were solved in ligand-
free forms and in the presence of 6-acetamido-6-deoxy-castanospermine (6-Ac-Cas). The E302Q variant was also trapped as an
intermediate with oxazoline bound to the active center. Crystallographic evidence highlights structural variations in the loop 3
environment, suggesting conformational heterogeneity for important active-site residues of this GH20 family member. The
enzyme was investigated for its β-N-acetylhexosaminidase activity toward chitooligomers and pNP-acetyl gluco- and galacto-
configured N-acetyl hexosaminides. Kinetic analyses confirm the β(1−4) glycosidic linkage substrate preference, and HPLC
profiles support an exoglycosidase mechanism, where the enzyme cleaves sugars from the nonreducing end of substrates. ScHEX
possesses significant activity toward chitooligosaccharides of varying degrees of polymerization, and the final hydrolytic reaction
yielded pure GlcNAc without any byproduct, promising high applicability for the enzymatic production of this highly valued
chemical. Thermostability and activation assays further suggest efficient conditions applicable to the enzymatic production of
GlcNAc from chitooligomers.
β-N-acetylhexosaminidases (HEX, E.C. 3.2.1.52) are glycosi-
dases that hydrolyze the glycosidic linkage of both gluco- and
galacto-configurations of N-acetyl-β-D-hexosaminides, which are
found in oligosaccharides, glycoproteins, glycolipids, and
glycosaminoglycans. HEX members have been extensively
studied due to their physiological and functional roles in
have also attracted considerable attention due to their
importance in human diseases; defects in human lysosomal
HEX causes neurodegenerative Tay-Sachs and Sandhoff
Received: December 23, 2013
Revised: February 19, 2014
Published: February 21, 2014
1
bacteria, fungi, insects, plants, and mammals. These enzymes
©
2014 American Chemical Society
1789
dx.doi.org/10.1021/bi401697j | Biochemistry 2014, 53, 1789−1800

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Scheme 1. Proposed Catalytic Mechanism of ScHEX (Based on Ref 1, See Text for Details)
2
19,20
diseases. Additionally, they have been used as a means to
chitinases act in a concerted fashion to degrade chitin fully.
control fungal and insect pests, in addition to being efficient
tools for a number of biotechnological applications. Based on
Previous genetic studies have revealed that, in the presence of
1
colloidal chitin and/or (GlcNAc) , S. coelicolor A3(2) secretes
2
amino acid sequence similarity, the Carbohydrate-Active
Enzyme Database (CAZy, http://cazy.org/) classifies HEX
members into four distinct families: GH3, GH20, GH84, and
GH85. The GH20 family has been thoroughly characterized
with respect to substrate specificity, structure, catalytic
mechanism, and biosynthetic potential. GH20 members have
the ability to cleave a broad range of substrates, including β(1−
chitinases to degrade chitin into chitooligomers, which are
subsequently hydrolyzed by HEX to produce GlcNAc in
2
1,22
vivo.
To date, numerous studies have focused on the
1
6,17,23−25
importance of S. coelicolor A3(2) chitinases,
but less
attention has been paid to the N-acetylhexosaminidase activity.
The goal of the present study was to clarify the chitinolytic
system of S. coelicolor A3(2) by providing detailed structural,
kinetics, and stability characterization of the β-N-acetylhex-
osaminidase activity from S. coelicolor A(3)2. Here, we report
the cloning and expression of the S. coelicolor A3(2)
SCC105.17c hex gene and the kinetic and structural character-
ization of the resulting protein. ScHEX possesses significant
activity toward chitooligosaccharides of varying degrees of
polymerization, and the final hydrolytic reaction yielded pure
GlcNAc without any byproduct, promising high applicability for
the enzymatic production of GlcNAc. Additional factors such as
enzyme preincubation with small saccharides also proved
efficient in activating enzyme efficiency.
1
4
), β(1−3), β(1−2), and/or β(1−6) glycosidic linkages, as well
as branched compounds such as glycoproteins, glycolipids, or
sulfated glycoconjugates. To date, a number of GH20 crystal
structures have been resolved, including bacterial, insect, and
3
−8
1
,9
human enzymes.
The catalytic domain of all members
presents a highly similar (β/α) -barrel (TIM-barrel) architec-
8
ture, with structural differences lying mainly in subunit
1
organization.
The catalytic mechanism of GH20 enzymes has also been
extensively investigated. The enzymes use a double-displace-
ment retaining mechanism with neighboring group participa-
1
tion (Scheme 1). In the first part of the reaction, the carbonyl
oxygen of the terminal 2-acetamido substrate moiety acts as a
nucleophile, yielding a bicyclic oxazoline (or oxazolinium ion as
shown in Scheme 1) intermediate, aided by a conserved
aspartate residue (D301 in ScHEX), and with protonic
assistance to leaving group departure provided by a conserved
acid/base (E302 in ScHEX). In the second step, the catalytic
base activates the incoming nucleophile (water, or another
sugar in the case of transglycosylation reactions) to form the
EXPERIMENTAL PROCEDURES
■
Reagents. All reagents used were of the highest commercial
purity available. Restriction enzymes and DNA-modifying
enzymes were purchased from New England Biolabs and
Roche Diagnostics. Chitooligosaccharides [(GlcNAc) ; n = 1−
] were purchased from V-Laboratories. pNP-N-acetyl-
galactosamine was purchased from Gold Biotechnology. 4-
Nitrophenyl N-N′-diacetylchitobioside [(GlcNAc) -pNP], 4-
nitrophenyl N-acetyl glucosaminide [(GlcNAc)-pNP], pNP-
pyranosides, and practical-grade crab shell chitin were
purchased from Sigma-Aldrich. Cell wall peptidoglycan from
the Gram-positive Bacillus subtilis and Streptomyces sp. were also
purchased from Sigma-Aldrich. Glycol chitin-80 (80% acety-
n
6
2
1
,10,11
reaction product.
GH20 enzymes have been used for the synthesis of
carbohydrates and glycomimetics in a number of biotechno-
logical applications. In particular, they can hydrolyze
chitooligomers generated from the chitin degradation process
to produce N-acetyl-D-glucosamine (GlcNAc), a monosacchar-
ide with significant added value in the medical and cosmetic
26
lated) was prepared as previously described. Chitosan-24
24% acetylated) was obtained from HaloSource. 6-Acetamido-
-deoxy-castanospermine (6-Ac-Cas) was purchased from
(
6
1
2
fields. In recent years, enzymatic processes have gained
efficiency at replacing harsh and environmentally damaging
GlycoSyn, Industrial Research Limited, New Zealand and di-
13
chemical processes for the industrial production of GlcNAc.
N-acetyl-chitobiose (GlcNAc) from Seikagaku Biobusiness,
2
‑
However, several issues remain with this production method,
which are mainly caused by product purity and productiv-
Japan. The strain S. lividans IAF 10-164 [msiK ] was used for all
27
protein expressions.
1
4,15
ity.
There is therefore impetus for investigating diverse
Cloning of ScHEX. All molecular biology procedures and
HEX enzymes that may result in more efficient production of
GlcNAc.
DNA manipulations in S. lividans were performed according to
28
standard published methods. The gene encoding for the β-N-
acetylhexosaminidase from Streptomyces coelicolor A3(2)
(SCC105.17c) was amplified by PCR using the following
DNA primers: 5′-AAAGCATGCGACCTCATCGACGGCAC-
CACAGAA-3′ (forward), coding for the ATG start codon
within a SphI restriction site (underlined), and 5′-
TTTGAGCTCTCAGGTCCAGGGCACCTG-3′ (reverse),
The Streptomyces species are well-known chitinolytic
organisms. Streptomyces coelicolor A3(2) is a Gram-positive
bacterium that produces many different hydrolases, including
1
6,17
chitinases and N-acetylhexosaminidases.
The sequenced
genome of S. coelicolor A3(2) reveals 13 putative chitinases and
18
1
1 putative HEX members. It is known that HEX and
1
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Table 1. Data Collection, Refinement, and Structure Quality Statistics for ScHEX and Complexes
ScHEX
ScHEX-6-Ac-Cas
E314Q-ScHEX-NGO
resolution of data (outer shell), Å
space group
52.24−1.85 (1.95−1.85)
P2 2 2
48.95−2.0 (2.10−2.0)
P2 2 2
41.35−1.80 (1.90−1.80)
P2 2 2
1
1
1
1
1
1
1 1 1
unit cell params
a = 72.7 Å
b = 128.9 Å
c = 150.3 Å
α = β = γ = 90°
0.064 (0.56)
16.8 (3.3)
99.9 (99.9)
6.1 (6.2)
120 786
0.17
a = 72.8 Å
b = 129.2 Å
c = 149.9 Å
α = β = γ = 90°
0.17 (0.53)
8.8 (3.2)
99.4 (98.6)
5.6 (5.7)
95 616
0.16
a = 58.2 Å
b = 64.5 Å
c = 143.7 Å
α = β = γ = 90°
0.10 (0.51)
12.2 (3.5)
100.0 (100.0)
7.1 (7.3)
50 954
0.18
a
R merge (outer shell)
mean I/sI (outer shell)
completeness (outer shell), %
multiplicity (outer shell)
no. unique reflns
Rcryst
Rfree
0.20
0.18
0.21
no. protein atoms
no. ligand atoms
no. solvent waters
7870
7847
3903
0
32 (6-Ac-Cas)
1150
14 (NGO)
456
904
b
no. EDO atoms
88
136
44
Rmsd for bonds (Å)
0.019
0.019
0.019
Rmsd for angles (°)
rmsd chiral volume (Å )
1.883
1.854
1.814
3
0.149
0.137
0.145
2
average protein B factor (Å )
27
11
19
2
average main chain B factor (Å )
average side chain B factor (Å2)
26
10
18
28
11
20
average B factor for ligand/EDO (ligand only) (Å2)
average solvent B factor (Å2)
43
22 (9)
24
28 (18)
29
39
PDB entry
4C7D
4C7F
4C7G
a
b
Rmerge = Σ Σ \ − <I >/Σ Σ \I . EDO is ethylene glycol.
hkl
i
Ihkli
hkl
hkl
i
hkli
coding for a TGA stop codon and a SacI restriction site
underlined). The SacI−SphI-digested PCR product was ligated
purity. Total protein concentration was determined using a Bio-
Rad Protein Assay (Bio-Rad).
(
into the 5.3 kb SacI−SphI-digested pC109 plasmid, which
carries a thiostrepton resistance marker. The resulting construct
was used to transform Streptomyces lividans 10-164 protoplasts
and plated on R5 medium without antibiotic for 16 h at 34 °C,
at which point agar plates were flooded with thiostrepton
solution and further incubated 3−4 days at 34 °C. The 6.9 kbp
plasmid was isolated, and positive clones were confirmed by
DNA sequencing. The E302Q mutation was introduced by
Mass Spectrometry. SDS-PAGE protein bands were
excised and destained with water/sodium bicarbonate buffer
and acetonitrile. Before in-gel digestion with trypsin, proteins
were reduced by DTT and alkylated with iodoacetamide. The
tryptic peptides were eluted and then separated on an Agilent
Nanopump using a trap column (ZORBAX 300 SB-C18) and a
reverse-phase column (ZORBAX 300 SB-C18, 150 mm × 75
μm, 3.5 μm particle size) (Agilent). Mass spectra were obtained
using a Q-trap mass spectrometer (Applied Biosystems/MDS
SCIEX instruments) equipped with a nanoelectrospray
ionization source. Accumulation of MS-MS data was performed
with the Analyst Software, version 1.4 (Applied Biosystems/
MDS SCIEX). MASCOT (Matrix Science) was used for
database searching.
29
overlap extension PCR using the following DNA primers: 5′-
AAAGCATGCCGCTACCTGCACATCGGCGGCGAC-
CAGGCGCACTCCACGCCGCAGGCC-3′ (forward) and 5′-
TTT GAGCT C GGCCTG CGG CG TG GAGT GCG-
GACCGTCGCCGCCGATGTGCAGGTAGCGGC-3′ (re-
verse).
Gene Expression and Protein Purification. Expression
Crystallization, Data Collection, and Structure Sol-
ution. Freeze-dried protein was solubilized in 10 mM CHES
buffer, pH 9.5, and crystallized using the hanging drop method,
with the drop containing ScHEX at 5 mg/mL in a ratio of 2.5: 1
with well solution (0.1 M HEPES pH 7.3, 8% (w/v) PEG
6000). The crystal was fished via a cryoprotectant, which
comprised the well solution supplemented with 25% (v/v)
ethylene glycol, into liquid nitrogen. Data were collected at
beamline IO3, Diamond, to 1.85 Å, at 100 K. Structure solution
of the ScHex gene was performed in S. lividans 10-164 using
28
slight modifications of previously published methods. Strains
were cultivated in 500 mL Erlenmeyer flasks for 72 h at 34 °C
with agitation (250 rpm) in M14 medium supplemented with
1
% xylose (w/v) as the sole carbon source. Mycelium was
removed by centrifugation, and the supernatant was filtered
through a 0.2 μm Whatman nylon membrane (GE Healthcare).
The protein solution was concentrated by ultrafiltration and
dialyzed against 10 mM citric buffer, pH 4.5 prior to loading
onto a Protein-Pak HR CM-8 AP-1 column (Waters) pre-
equilibrated with 10 mM citric buffer, pH 4.5. ScHEX was
eluted with a linear gradient of 10 mM citric buffer, pH 4.5
containing 1 M NaCl, and protein elution was monitored at
30
used the CCP4 suite of programs. The structure was solved
31
by Molecular Replacement with PHASER, using PDB entry
10
1HP4 as a model (94% sequence identity). The model was
32
improved by iterative cycles of manual building in COOT,
followed by refinement with REFMAC.
33
280 nm. Fractions containing ScHEX were pooled after analysis
For the complex with 6-acetamido-6-deoxy-castanospermine
by SDS-PAGE, and mass spectrometry was used to assess
(6-Ac-Cas), a crystal which had grown after 4 weeks in a
1
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Biochemistry
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hanging drop, comprising protein at approximately 7 mg/mL in
purified enzyme was added to a final concentration of 10 μg/
mL. Reactions were performed for 24 h with aliquots taken
after 18h and 24h of incubation at 55 °C. Products were
detected at 210 nm by an isocratic HPLC ion exclusion method
using an Aminex HPX-42A column (7.8 mm × 30 cm) (Bio-
5
0 mM HEPES pH 7.5, 0.2 M NaCl, 20 mM CHES pH 9.3,
mixed 1:1 with well solution consisting of 0.1 M BIS-TRIS pH
.5, 4% (w/v) PEG 6000, 5% DMSO, was soaked with a speck
6
of solid 6-Ac-Cas introduced around the crystal with a needle.
Following soaking for approximately 40 min, the crystal was
fished via cryoprotectant (as above). Data were collected at
beamline IO2, Diamond, to 2 Å resolution, although the crystal
was of poor quality reflected by a relatively high Rmerge. The
structure was refined using the (isomorphous) wild-type
structure as the starting model, using REFMAC and COOT.
A crystal of the site-directed mutant E302Q, initially in
complex with the disaccharide di-N-acetyl-chitobiose
Rad). Elution was performed with H O as the mobile phase at a
2
flow rate of 0.5 mL/min (45 °C). In all cases, control assays
were also performed with water and a mock supernatant.
Optimal pH, Temperature, and Stability. Optimal
temperature for ScHEX was determined in 50 mM phosphate
buffer containing 150 mM NaCl with chitooligosaccharides
[(GlcNAc) ; n = 1−6] as substrates. Enzyme assays were
n
performed for 10 min at 25, 30, 37, 40, 45, 50, 55, 60, 65, and
70 °C. The optimal pH for chitooligosaccharide hydrolysis was
determined at 55 °C by performing the same enzyme assays in
50 mM citric buffer with 150 mM NaCl (pH 3, 3.5, 4, 4.5), 50
mM phosphate buffer with 150 mM NaCl (pH 5, 5.5, 6, 6.5, 7),
and 50 mM Tris-HCl buffer with 150 mM NaCl (pH 8).
Reactions were carried out for 10 min, and enzyme activity was
calculated as mentioned above. The pH stability and thermal
stability of ScHEX were estimated by incubating enzyme
solutions (100 μL, 200 μg/mL) at different pHs (1h and 3
months at 4 °C) and temperatures (15 min) prior to initiating
enzyme reaction with pNP-GlcNAc, as described above.
Activity was compared to a control, which was performed
with pure enzyme kept in water at 4 °C.
(
GlcNAc) , was crystallized at 5 mg/mL in 10 mM CHES
2
pH 9.5, mixed 1:0.8 with well solution comprising 0.1 M
HEPES pH 7.0, 4% (w/v) PEG 6000. A small amount of solid
(
GlcNAc) powder was added to the protein drop, and the
2
crystal was fished after 5 days via 6% (w/v) PEG 6000, 0.1 M
HEPES pH 7.0, 25% (v/v) ethylene glycol also supplemented
with 20 mM (GlcNAc) . Data were collected at beamline IO4
2
Diamond, to 1.8 Å resolution. The structure was solved using
PHASER, with the wild-type structure as the search model and
refined as above. Upon structure solution it became
immediately apparent that the ligand bound was the trapped
intermediate GlcNAc (NAG)−oxazoline (NGO) rather than
the intact substrate (see below). Data collection and structure
refinement statistics are given in Table 1.
β-N-Acetylhexosaminidase Activity. β-N-acetyl-
hexosaminidase activity against p-nitrophenyl β-N-acetyl-
hexosaminides and p-nitrophenyl pyranosides was determined
by continuous spectrophotometric assays by measuring the
release of p-nitrophenol at 405 nm on a Cary 300 Bio
spectrophotometer (Agilent) equipped with a circulating
heating water bath (Lauda). Reactions were carried out at 55
Michaelis−Menten Kinetics. The kinetic parameters kcat
and K were determined at 55 °C and optimal pH in 50 mM
M
phosphate buffer, 150 mM NaCl for chitooligosaccharide
substrates [(GlcNAc)
; n = 1−6]. Enzyme assays were initiated
n
by the addition of 0.5 μg/mL ScHEX with substrate
concentrations ranging from 0.25 to 3 mM. ScHEX activity
was calculated as μmoles of GlcNAc released per minute per
milligram of enzyme, and kinetic parameters k , K and k /
cat
M
cat
KM were calculated by a nonlinear regression fit to the
Michaelis−Menten equation using GraphPad Prism 5.
Carbohydrate Activation/Inhibition. The effect of
potential activators and inhibitors on β-N-acetylhexosaminidase
activity was examined by preincubating enzyme solutions (100
μL, 200 μg/mL) with 3 mM of the following carbohydrates for
°C for 10 min in 50 mM phosphate buffer containing 150 mM
NaCl (pH 5.5). Activity was expressed in IU/mg of protein (1
IU = 1 μmole of p-nitrophenol released per minute).
Determination of ScHEX enzyme activity toward chitooligo-
saccharides was performed by an isocratic HPLC method in the
same buffer conditions. Aliquots (100 μL) were taken at various
time intervals and mixed with 100 μL of 0.4 M H SO to
1
5 min prior to reaction initiation: mannose, fructose,
2
4
arabinose, glucuronic acid, sucrose, galactose, glucose, xylose,
lactose, and GlcNAc. Enzyme assays were carried out with
pNP-GlcNAc as described above, and sugars were prepared in
the same reaction buffer. Activities were compared to a control
of pure ScHEX performed under the same reaction conditions
but preincubated in the absence of any sugar.
quench the reaction. Samples (100 μL) were subsequently
injected on an Aminex HPX-87H Ion Exclusion Column (7.8
mm × 30 cm) (Bio-Rad), and elution was performed in
isocratic mode with a mobile phase of 5 mM H SO at a flow
rate of 0.5 mL/min (45 °C). Identification of molecular species
was performed by comparison with the retention time of
known standards, and product concentrations were calculated
from HPLC peak areas. Activity was expressed in IU/mg of
protein (1 IU = 1 μmole of GlcNAc released per minute). The
growth supernatant of a pC109-transformed S. lividans 10-164
subjected to the same gene expression protocol was used as a
mock control for all enzyme assays.
2
4
RESULTS AND DISCUSSION
■
ScHEX Expression, Purification, and Structural Anal-
ysis. ScHEX was produced from a culture of S. lividans grown
in M14 minimal medium containing xylose as the sole source of
carbon. Under these conditions, ScHEX was overproduced with
an expression yield of 700−800 mg/L after 72 h incubation.
The S. lividans expression system allows extracellular secretion
of ScHEX with an extremely low background of contaminant
proteins, yielding an enzyme purity of 95% directly from the
growth supernatant. Ion-exchange chromatography was never-
theless employed to purify enzymes to homogeneity, and mass
spectrometry was employed to confirm the identity and
sequence integrity of the produced enzyme. To rule out the
possibility of a contaminant GH activity, the strain S. lividans
10-164 with empty plasmid pC109 was cultured to test the
Substrate Specificity. Substrate specificity of the purified
ScHEX was determined using chromogenic compounds pNP-
GlcNAc, pNP-GalNAc, and pNP-pyranosides, in addition to
natural chitins and chitooligosaccharides of varying degrees of
polymerization [(GlcNAc) ; n = 1−6]. To investigate the
n
activity toward long chain chitins, enzyme assays were carried
out in the aforementioned buffer using either 2.5 mg/mL of
80% glycol chitin, 40% glycol chitin, 24% chitosan, chitin
crabshell, colloidal chitin or 1 mg/mL peptidoglycan from
Streptomyces sp. or Bacillus subtilis. To initiate the reaction, the
1
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Figure 1. Structural comparison between SpHEX and ScHEX. (A) Front-view overlay of wild-type SpHEX (PDB entry 1HP5, red) with wild-type
ScHEX (PDB entry 4C7D, cyan). The position of the active site is defined by the presence of NAG−thiazoline bound within the SpHEX binding
pocket (NGT, yellow). The β/α topology domain I and (β/α) barrel domain II are identified by roman numerals. Residues of the (β/α) barrel
8
8
comprising catalytic domain II that differ in sequence between SpHEX and ScHEX are shown in ball-and-stick representation in ScHEX (blue). (B)
Side-view of the complex (90° counter clockwise rotation about the y-axis relative to (A)). (C) Stereo view of SpHEX (PDB entry 1M01, red) and
ScHEX (cyan/green) showing two alternate conformations adopted by loop 3 in ScHEX. Positions of the modeled loop 3 conformations A (0.7
occupancy) and B (0.3 occupancy) are shown in green and red, respectively. The loop 3B conformation corresponds to the active orientation of
catalytic residues Asp301B and Glu302B (cyan in ScHEX and red in SpHEX), which are ideally positioned to hydrogen bond with glycans in the
active-site pocket. Residues Asp301A and Glu302A in conformation A are shown in green. The N-acetylglucosamine (GlcNAc) product complexed
to SpHEX is shown in yellow. This figure was drawn with MacPyMOL 1.3.
Figure 2. Stereo view of a ribbon diagram of ScHEX. The enzyme is color-ramped from the N-terminus (blue) to C-terminus (red), showing the
5
2
position of 6-Ac-Cas as a CPK model. This figure was drawn with CCP4mg.
growth supernatant for acetylhexosaminidase activity against
sequence identity with S. lividans, S. plicatus, S. avermitilis,
Saccharopolyspora erythraea, Paenibacillus sp., and Homo sapiens
(HEXB), respectively (Figure S1). Like other members of the
family, ScHEX retains the highly conserved active site motif
(
GlcNAc) and (GlcNAc) . Results showed no activity toward
2 6
these substrates, ruling out the possibility of a contaminant
HEX activity in the cell extract of the expression host.
Amino acid sequence alignment of ScHEX with other GH20
family members exhibits 99%, 94%, 77%, 60%, 43%, and 27%
His/Asn-X -Gly-Ala/Cys/Gly/Met-Asp-Glu-Ala/Ile/Leu/Val,
aa
in which a glutamate residue (E302 in ScHEX) acts as the
1
793
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Figure 3. Stereo views (divergent “wall-eyed” stereo) of the ScHEX 6-Ac-Cas and E302Q ScHEX NAG−oxazoline active-site complexes. F -F omit
o
c
electron density maps (with phases calculated prior to any modeling or refinement of any ligand) of each complex are shown in blue, contoured at
.0 σ. (A) ScHEX 6-Ac-Cas complex (molecule A). (B) E302Q ScHEX NAG−oxazoline complex. Hydrogen bonds ≤3.2 Å are shown as dashed
lines. This figure was drawn with CCP4mg.
3
3
4
general acid/base in the catalytic mechanism. The closest
HEX homologue for which a crystal structure was previously
solved is the GH20 β-N-acetylhexosaminidase from S. plicatus
residue 302 are hydrogen bonded to NE2 of His238, but in the
native structure, molecule A and conformation A of region
299−308 on molecule B, the side chain of Glu302 points away
from, and cannot hydrogen bond to the histidine. This alternate
ligand-bound conformation facilitates the formation of hydro-
gen bonding interactions. In the 6-Ac-Cas structure, OD2 of
Asp301 is hydrogen bonded to the nitrogen of the ligand
acetamido group (2.8 Å), and OE2 of Glu302 is 2.8 Å from the
castanospermine nitrogen atom (Figure 3A). In the complex
with NAG−oxazoline, the loop is positioned in such a way that
there are hydrogen bonds between OD2 of Asp301 and the
nitrogen atom of NAG−oxazoline (2.9 Å), and between OE1 of
Gln302 and O3 of NAG−oxazoline (2.8 Å) (Figure 3B). For
molecule B of the native structure, the alternate loop
conformations differ in some main chain hydrogen bonding
interactions, for example the carbonyl O of Asp301B loop in
conformation A is hydrogen bonded to the main chain N of
Gly237B, whereas in loop B it is hydrogen bonded to that of
Ala303B (not shown).
(
SpHEX), an enzyme displaying 94% sequence identity with
1
0
ScHEX. As expected, this high similarity between the two
enzymes results in very similar three-dimensional structures,
discussed below. This is in agreement with the fact that the
catalytic domains of SpHEX and ScHEX only differ at 21 out of
3
55 residue positions. None of these replacements occur at
positions in or near the active site cavity and/or involved in
catalysis (Figure S1, Figure 1A,B), confirming that both
enzymes rely on the same catalytic mechanism and display
similar substrate specificities.
Structure of ScHEX, and Complexes ScHEX-6-Acet-
amido-6-deoxy-castanospermine and E302Q ScHEX-
NAG−oxazoline. The structure of ScGH20 has a very similar
two domain architecture to that of SpHEX, with an N-terminal
domain (up to Arg138) consisting predominantly of a solvent-
exposed seven-stranded β-sheet, sequestering two α-helices
against the C-terminal domain, which comprises a (β/α) barrel
In an attempt to trap a Michaelis complex, the crystal
structure of the acid/base variant E302Q was determined in
complex with di-N-acetyl-chitobiose. In contrast to what was
expected, the structure clearly revealed electron density for a
trapped intermediate oxazoline in the −1 subsite. In trapping of
oxazolines on related GH84 enzymes, He and colleagues used
active center variants in conjunction with highly activated
substrates; here serendipitously the intermediate has accumu-
lated without the requirement for an activated substrate. As
expected, the NAG−oxazoline and active site residues occupy
very similar positions to those in the complex of the inhibitor
N-acetylglucosamine−thiazoline (NAG−thiazoline) with
SpHEX (PDB entry 1HP5).
8
with an additional α-helix near the C terminus occupying the
10
groove between the two domains (Figure 2). The barrel motif
is irregular, in that helix 7 is replaced by a loop, helix 5 is very
short, and there are 2 additional loops at the C-termini of β-
strands 2 and 3. There are two molecules in the asymmetric
unit for the native structure, and the complex with 6-Ac-Cas,
designated A and B in the PDB entries, and one for that of the
35
mutant E302Q soaked with (GlcNAc) , which has NAG−
2
oxazoline in the active site.
In molecule A of the native structure, residues 299−308 on
loop 3 follow a different pathway to the equivalent residues in
both complex structures (Figure 1C). In molecule B, the same
region has two alternate conformations, A and B, with partial
occupancies of 0.7 and 0.3, respectively. Conformation A is the
same as that observed for molecule A, and loop conformation B
is equivalent to that observed in both ligand-bound structures
and the structure of SpHEX bound to GlcNAc (PDB entry
The trapped oxazoline complex, along with the complex with
6-Ac-Cas informs a complete description of the interaction in
4
the −1 subsite. Both ligand sugars are close to a C1
conformation. The acetamido group is anchored by hydrogen
bonding interactions between its nitrogen atom and OD2 of
Asp301, and oxygen and OH of Tyr381 (Figure 3, Scheme 2).
The structure of GlcNAc bound to SpHEX exhibits a similar
pattern of bonding between the equivalent N and O atoms of
1
1
1
M01). The main difference is in the orientations of the side
chains of Asp301, Glu302 (both catalytic residues) and Ala303.
In conformation B, and both complexes, the side chains of
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Scheme 2. Schematic Diagram of the Interactions between
which lies 2.7 Å from the side chain of Gln302. The wild-type
structure also has an ethylene glycol molecule (at half
occupancy) in a different orientation, but with a hydroxyl
group in a position equivalent to the hydroxyl binding to
Gln302 in the NAG−oxazoline structure, and a second
ethylene glycol with hydroxyl groups occupying similar
positions to NAG−oxazoline O6 and O4.
ScHEX and the Intermediate Oxazoline Trapped on the
a
E302Q Variant
Enzyme Specificity. Glycoside hydrolases are classified
into 130 families based on their amino acid sequence
36−38
similarities.
Although members of the GH20 family
share a common catalytic domain with conserved active site
residuesconfirmed by the detailed description of the
structure abovethey nevertheless show a wide variety of
substrate specificities. Overall, GH20 enzymes have the ability
to cleave β(1−2), β(1−3), β(1−4), β(1−6) glycosidic linkages
from oligosaccharides, glycoproteins, glycolipids, and glyco-
a
Hydrogen bonding distances ≤3.2 Å are indicated by dashed lines.
8,39−41
saminoglycans.
The GH20 family also relies on a
The water molecule is shown as a shaded sphere.
substrate-assisted mechanism wherein the 2-acetamido group
of the substrate is indispensable for catalysis (Scheme
11
1
,10,11,42
the ligand and residues Asp313 and Tyr393. Four tryptophan
residues, 332, 349, 396 and 430, provide a hydrophobic
environment that protects the oxazolinium ion intermediate
from solvolysis (Figure 4). The positive charge on the nitrogen
of the acetamido group in the NAG−oxazoline complex is
likely stabilized by Asp301, which is itself within hydrogen
bonding distance of Asp234, and therefore probably deproto-
nated. The oxygen atom O3 of NAG−oxazoline is 2.8 Å from
OE1 of the mutated residue Gln302, and in the 6-Ac-Cas
complex N1 of the castanospermine group is hydrogen bonded
to OE2 of Glu302 (2.8 Å). Further residues interact, via
hydrogen bonding interactions with their side chains, to hold
the sugar moiety of both ligands in place as follows: Asp179
1
).
To determine the substrate specificity of ScHEX
and to confirm its anchimeric mode of action, we tested
chromogenic and natural substrates with 2-acetamido groups
such as pNP-GlcNAc, pNP-GalNAc, chitooligosaccharides, and
long chain chitins. As expected, the enzyme showed significant
activity toward pNP-GlcNAc, pNP-GalNAc, and chitooligosac-
charides (Table 2). The highest specific activity (666 ± 11 IU/
Table 2. Specific Activity of ScHEX toward Various N-
Acetylhexosaminide Substrates
a
substrate
specific activity (IU)
b
pNP-GlcNAc
pNP-GalNAc
(GlcNAc)6
(GlcNAc)5
(GlcNAc)4
(GlcNAc)3
(GlcNAc)2
666 ± 11
230 ± 4
b
(
OD2 via a water molecule to O4 of 6-Ac-Cas and O3 of
NAG−oxazoline), Arg150 (both terminal guanidinium N
atoms with O4 of 6-Ac-Cas, and one each to O3 and O4
NAG−oxazoline), Glu432 (OE2 to O2 of 6-Ac-Cas and O4 of
NAG−oxazoline) and Asp383 and Trp396 (OD2 and NE1
respectively via a water molecule to O2 of 6-Ac-Cas, directly to
O6 of NAG−oxazoline) (Figure 3, Scheme 2). In addition, a
water molecule, which is hydrogen bonded to both OD2
Asp301 and OD2 Asp179, forms a hydrogen bond to O4 6-Ac-
Cas and to O3 NAG−oxazoline in their respective complexes
180 ± 25
146 ± 11
71 ± 6
122 ± 12
126 ± 5
a
b
IU = μmol/min of GlcNAc released per milligram of enzyme. IU =
μmol/min of pNP released per milligram of enzyme. Results are
presented as means ± standard deviation (n = 3).
(
3.0 Å in each case).
mg) was observed with pNP-GlcNAc as substrate, which is 2.9-
fold higher than that observed with the homologous pNP-
GalNAc (230 ± 4 IU/mg). This result confirms the D-gluco
configuration substrate preference of ScHEX, in agreement with
the 1.5 to 4 GlcNAcase/GalNAcase activity ratio found in other
In the SpHEX NAG−thiazoline complex, a glycerol molecule
occupies subsite +1 and forms a hydrogen bond with OE2
Glu314 (2.7 Å). There is an equivalent ethylene glycol
molecule in the E302Q ScHEX NAG−oxazoline structure,
Figure 4. Stereo view of hydrophobic residues in the −1 sugar binding subsite for molecule A of ScHEX complexed with 6-Ac-Cas. The position of
NGO is shown when the structure of its complex is superposed on that with 6-Ac-Cas. 6-Ac-Cas is shown with C atoms in green, and NGO is shown
in coral. The molecular surface around the hydrophobic residues is shown (calculated by CCP4mg).
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1
9
GH20 enzymes. ScHEX also displayed specific activities
ranging from 71 to 180 IU/mg toward chitooligosaccharide
substrates (Table 2), confirming that the enzyme most likely
acts on smaller chain products of chitin degradation in vivo. To
further investigate the glycosidase activity, time-course profiles
for the hydrolysis of (GlcNAc)6 were determined under
conditions described above. Time-course analysis of the
the nonreducing ends of substrates of varying degrees of
polymerization, as previously demonstrated on homo-
3
,5,43
logues.
The same cleavage pattern was observed when
(GlcNAc)2−5 chitooligomers were used as substrates, with the
appearance of n−1 products at an early stage of the reaction,
concomitant with gradual increases in GlcNAc concentrations
(not shown).
The ScHEX activity was also tested against longer chain
chitins and bulkier substrates, including glycol chitin 80%,
glycol chitin 40%, chitosan 24%, chitin crabshell, colloidal
chitin, and peptidoglycans from Bacillus subtilis and Streptomyces
sp. After 18 and 24 h of incubation, no significant oligomer was
detected in the reaction mixture, except for very low
concentrations of GlcNAc (not shown). GlcNAc was not
detected in identical assays conducted with the mock
supernatant or water as negative controls, confirming that the
activity is specific to ScHEX. The bulk of these observations
indicate that ScHEX possesses N-acetylhexosaminidase activity
toward short chain chitin oligomers but not toward long chain
chitin or chitosan. This result was expected given the high
sequence identity and similar activity observed with other
GH20 family members, namely the highly homologous
ScHEX activity toward (GlcNAc) yielded a hydrolysis profile
6
whereby (GlcNAc)5 and GlcNAc are released as primary
products at the early stage of the reaction, followed by
decreasing concentrations of (GlcNAc) , (GlcNAc) , and
4
3
(
GlcNAc) (Figure 5). The corresponding increase in GlcNAc,
2
the low accumulation of smaller oligomers such as (GlcNAc)2
at the early onset, and the very similar affinity of ScHEX for all
chitooligosaccharides (see below) confirms that the enzyme
acts in a processive manner, trimming off GlcNAc units from
44
SpHEX. Similar to most other GH20 members characterized,
increasing the length and complexity of chitin substrates
considerably affects enzyme affinity and catalysis in ScHEX.
Michaelis−Menten Kinetics. ScHEX showed lower
specific activity in its purified form than that calculated from
the fresh outer-cell growth supernatant immediately after
protein expression, most likely due to poor enzyme stability
in solution over time. As a result, kinetic parameters were
calculated from the 95% pure ScHEX, providing a better
portrait of the in vivo activity of ScHEX. Considering the high
purity of the enzyme in the outer-cell medium and the absence
of background activity from the expression organism (as tested
from a control S. lividans growth assay), this expression system
provides a considerable advantage for the industrial production
of ScHEX. Indeed, protein purification often cannot be
performed in industrial settings due to high costs, setup and
productivity issues. The kinetic parameters KM and kcat were
calculated for each chitooligomer under known optimal
conditions (see below). Hydrolysis of the substrates showed
typical Michaelis−Menten hyperbolic kinetics with substrate
concentrations ranging from 0.25 mM to 3 mM (Figure S2).
Results show that K and k parameters are very similar for all
M
cat
substrates, varying from a mere 2- to 3-fold difference in all
cases (Table 3). The enzyme displays the lowest affinity toward
Table 3. Kinetic Parameters for ScHEX
kcat (s−1)
k /K (mM
−1 −1
s
)
substrate
KM (mM)
cat
M
(GlcNAc)2
(GlcNAc)3
(GlcNAc)4
(GlcNAc)5
(GlcNAc)6
1.97 ± 0.42
0.76 ± 0.10
0.90 ± 0.15
0.59 ± 0.09
0.98 ± 0.22
593 ± 94
330 ± 16
232 ± 10
271 ± 1
297 ± 12
432 ± 25
258 ± 20
459 ± 19
423 ± 63
409 ± 63
Figure 5. Time-course hydrolysis of (GlcNAc) by ScHEX. Samples
6
were analyzed by measuring absorption at 210 nm following an
isocratic HPLC separation of products on an Aminex HPX-87H ion
exclusion column at 45 °C, as described in Experimental Procedures.
Chromatograms are shown for acid-quenched reactions after 0, 4, 6,
(
GlcNAc) (K = 1.97 ± 0.42), which correlates with a slightly
2 M
higher kcat (kcat = 593 ± 94). However, these differences do not
translate into higher catalytic efficiency of ScHEX for this
substrate and no other significant catalytic efficiency differences
were observed for substrate chain lengths ranging from 2 to 6
N-acetylglucosamine units [(GlcNAc)2−6]. These results
demonstrate that substrate affinity is very similar for short
10, 20, and 30 min of incubation. Retention times were compared to
known chitinooligosaccharide standards: VI, (GlcNAc) ; V,
6
(
(
GlcNAc) ; IV, (GlcNAc) ; III, (GlcNAc) ; II, (GlcNAc) ; I,
GlcNAc); HPr, propionic acid normalization standard.
5 4 3 2
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Figure 6. Effect of pH and temperature on ScHEX activity against chitooligosaccharides. (A) Effect of temperature on ScHEX activity. Enzyme
activity was tested for (GlcNAc) (blue squares), (GlcNAc) (green up triangles), (GlcNAc) (red down triangles), (GlcNAc) (purple diamonds),
2
3
4
5
and (GlcNAc) (black circles). (B) Effect of pH on ScHEX activity (same symbols and color code). (C) Residual activity of ScHEX against pNP-
6
GlcNAc after a 15-min incubation at various temperatures. (D) Residual activity of ScHEX against pNP-GlcNAc after 1 h (black squares) and 3-
month (red up triangles) incubation at various pHs. See Experimental Procedures for details.
chitooligomers of varying degrees of polymerization, supporting
the observation that ScHEX is an exo-acting glycosidase that
cleaves from the nonreducing end of substrates through a
processive hydrolytic mechanism, as previously demonstrated
temperatures ranging from 45 to 60 °C with a maximal activity
observed at 55 °C for almost all substrates tested. These values
are similar to those observed in GH20 members from Sotalia
7
45
46
fluviatilis, Streptomyces thermoviolaceus, Penicillium oxalicum,
3
,5,43
47
on homologues.
and Aspergillus oryzae. Enzyme activity toward (GlcNAc) was
6
Most GH20 members have been kinetically characterized in
the presence of unnatural substrate analogues, such as pNP-
GlcNAc, pNP-GalNAc, and/or 4-methylumbelliferyl-β-D-gal-
actopyranosides (4-MUG). Only a restricted set of HEX
enzymes has been studied in the presence of natural
particularly affected outside the 55−60 °C range (Figure 6A),
dropping to near zero at physiological temperatures or lower.
The smaller substrates (GlcNAc)2−4 were not as affected by
drastic temperature drops as (GlcNAc)5−6, further lending
support to the biological importance of ScHEX in hydrolysis of
small chitooligosaccharides in the chitinolytic system of S.
coelicolor A(3)2.
49
chitooligomers, including ExoI from Vibrio furnissii, Le-
53
Hex20A from Lentinula edodes, VhNag from Vibrio harveyi
4
3
54
6
50, and StmHex from Stenotrophomonas maltophilia. The
Thermal stability of ScHEX was also assessed by character-
izing enzyme stability after a 15 min incubation at temperatures
ranging from 25 to 70 °C prior to reaction initiation (Figure
6C). Despite an optimal activity of 55−60 °C, ScHEX stability
at higher temperatures was considerably reduced, dropping
dramatically for temperatures higher than that of the optimal
growth of its host organism (30 °C). Indeed, while ScHEX
retained 43% activity at 50 °C, this efficiency dropped to less
than 5−6% when incubated at higher temperatures. This
property is similar to NagA from Pseudomonas fluorescens JK-
catalytic efficiency of ScHEX compares advantageously with
other HEX members, showing very similar k /K against
cat
M
(
GlcNAc)2−6 substrates. In fact, ScHEX from crude supernatant
is 1700-fold more efficient than pure StmHex against
5
4
(
GlcNAc)6. Still, even with equal catalytic efficiency, ScHEX
remains advantageous from an industrial standpoint due to its
chitin degrading ability in the growth supernatant, thus
preventing the need for further protein purification.
Enzyme Stability. To provide details on the potential
applicability and robustness of ScHEX for the industrial
production of GlcNAc, the effect of temperature on activity
was determined by monitoring hydrolysis of (GlcNAc)2−6
chitooligosaccharides from 25 to 70 °C (Figure 6A). Although
Streptomycetes are soil-dwelling bacteria that thrive at
mesophilic temperatures, ScHEX showed optimal activity at
5
48
0412 and β-HEX from Capsicum annuum. These results
demonstrate the in vivo trade-off between enzyme stability and
catalytic efficiency, whereby increased stability at lower
temperatures is favored over increased enzyme activity. While
far from a catalytically perfect enzyme, ScHEX is just good
enough to perform its required chitinolytic activity in vivo,
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providing S. coelicolor A(3)2 with sufficient enzymatic activity
to survive.
galactose do not significantly affect ScHEX activity, both sugars
were shown to increase chitinohydrolysis of the Sotalia
fluviatilis enzyme by 43% and 80%, respectively. Interestingly,
preincubation of ScHEX with the reaction product GlcNAc
increased enzyme activity by more than 35% relative to control,
indicating that product inhibition is not an issue with ScHEX,
contrasting with results observed with other GH20 members,
namely, PoHEX and ExoI from Penicilium oxalicum and Vibrio
The effect of pH on ScHEX activity was also determined by
monitoring the hydrolysis of (GlcNAc)2−6 chitooligosacchar-
ides from pH 3 to 8 (Figure 6B). ScHEX showed a broad pH
tolerance, with optimal activity detected between pH 4.5 and 7.
Again, (GlcNAc)6 proved a more stringent substrate by
displaying optimal activity between pH 5 and 6.5. Many
other GH20 enzymes, such as HEX from Sotalia fluviatilis,
NagC from Streptomyces thermoviolaceus, HEX1 and HEX2
from Paenibacillus sp. TS12, and DspB from Aggregatibacter
actinomycetemcomitans, showed similar optimal activity between
7,49
furnissii, respectively.
Overall, the activating effects of
fructose and glucuronic acid on ScHEX activity are significant
and could be used to enhance the efficiency of the enzyme in
GlcNAc production. The exact molecular phenomenon under-
lying such activation was never investigated in the GH20 family
but could result from a potential induced fit and/or
conformational selection mechanism triggered by the binding
of substrate analogues, thus favoring a catalytically competent
enzyme-bound state over the unbound, inactive ground state.
This type of ligand binding mechanism was previously
characterized on a number of other enzyme systems and has
recently attracted considerable attention because both mech-
anisms may act as a flux and be affected by changes in ligand
4
,39,45,46
pH 5 and 6.
Interestingly, ScHEX did not display
significant pH-dependent activity variations between chitoo-
ligomers of varying degrees of polymerization. This result
contrasts with previous observations on β-GlcNAcase and
chitodextrinase from Vibrio furnissii, two enzymes that showed a
shift in pH-activity profiles with the smaller (GlcNAc)
substrate relative to longer chitin oligomers.
2
49
ScHEX also displayed very good residual activity after
incubating the enzyme at different pH values for extended
periods of time (1 h and 3 months) (Figure 6D). The enzyme
maintained almost the same level of activity between pH 4−8
after 1 h or 3 months of incubation in different pH buffer
conditions. Additionally, although enzyme activity was
significantly affected after 3 months of incubation at 4 °C,
ScHEX retained the same residual pH-activity profile as that of
the fresh enzyme.
Effect of Carbohydrates on ScHEX Activity. To
elucidate the effect of possible activators/inhibitors on catalytic
efficiency, ScHEX was preincubated for 15 min in the presence
of 3 mM of the following sugars prior to reaction initiation:
mannose, fructose, arabinose, glucuronic acid, sucrose,
galactose, glucose, xylose, lactose, and GlcNAc. Interestingly,
mannose, fructose, glucuronic acid, lactose, and GlcNAc
significantly increased enzyme activity by a factor of 33%,
50
concentrations.
CONCLUSION
■
In the present work, we found that the β-N-acetyl-
hexosaminidase from Streptomyces coelicolor A3(2) can be
produced extracellularly in high yield and with good purity
from S. lividans cultures. The 3D structure of ScHEX is very
similar to that of SpHEX, with which it shares 94% sequence
identity. Complexes have been obtained with the inhibitor 6-
Ac-Cas and by soaking a catalytic acid/base residue variant of
the enzyme, E302Q, with chitobiose. This allowed the capture
of the NAG−oxazoline intermediate in the −1 subsite, which
binds in a similar manner to the stable inhibitor NAG−
thiazoline. The enzyme extract exhibits high chitinolytic activity
toward chitooligosaccharides, offering potential for the
production of GlcNAc as a chemical precursor. The enzyme
demonstrates a wide pH-activity range from 4.5 to 7 and
exhibits optimal activity at temperatures up to 55 °C. Kinetic
measurements for ScHEX with chitin oligomers showing
degrees of polymerization 2−6 have shown k /K values of
47%, 63%, 28%, and 35%, respectively (Table 4). In contrast, β-
Table 4. Effect of Carbohydrates on ScHEX Activity
a
compd
control
relative activity (%)
cat
M
100
a similar order of magnitude. These kinetic parameters are
superior or comparable to previously described β-N-acetyl-
hexosaminidases, offering an improvement for the industrial
application of ScHEX. The release of sequentially shorter chitin
mannose
fructose
arabinose
glucuronic acid
sucrose
133 ± 5.3
147 ± 8.2
128 ± 32
163 ± 10
82 ± 12
83 ± 12
104 ± 13
92 ± 12
128 ± 14
135 ± 15
oligomeric products from (GlcNAc) over time lends support
6
to an exoglycosidase activity, with the enzyme cleaving sugars
from the nonreducing end of substrates, as with other GH20
family members. Enzyme activity can be significantly stimulated
in the presence of sugars, notably fructose, glucuronic acid, and
GlcNAc. Thus, some sugars may be used to increase yield and
product inhibition is not an issue. Further work will enable the
identification of conditions that can maximize the potential
yield of GlcNAc by such an extract in an industrial setting.
ScHEX adds to the GH20 repertoire, from a structural and
mechanistic standpoint and provides an enabling technology for
the high-level bioproduction of monosaccharides.
galactose
glucose
xylose
lactose
GlcNAc
a
Enzyme assays were carried out with 0.25 mM pNP-GlcNAc using
ScHEX preincubated for 15 min with 3 mM of each compound.
Activities were compared with control, which was performed with pure
ScHEX. Results were presented as means ± standard error (n = 3).
N-acetylhexosaminidase activity was either unaffected or weakly
inhibited by arabinose, sucrose, galactose, glucose, and xylose.
The stimulating effect on HEX activity by sugars was also
observed in HEX from Sotalia fluviatilis, which displayed
comparable increases upon incubation with glucuronic acid
ASSOCIATED CONTENT
Supporting Information
A sequence alignment of ScHEX with members of the GH20 β-
N-acetylhexosaminidase family and Michaelis−Menten kinetics
profiles for the hydrolysis of (GlcNAc)2 by ScHEX. This
■
*
S
45
(
65%), fructose (58%), and lactose (70%). While sucrose and
−6
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material is available free of charge via the Internet at http://
pubs.acs.org.
acetylhexosaminidase by comparative molecular modeling and site-
directed mutagenesis. J. Biol. Chem. 273, 19618−19624.
(7) Ryslava, H., Kalendova, A., Doubnerova, V., Skocdopol, P.,
Accession Codes
Coordinates and structure factors have been deposited in the
Protein Data Bank with accession numbers 4C7D, 4C7F, and
Kumar, V., Kukacka, Z., Pompach, P., Vanek, O., Slamova, K.,
Bojarova, P., Kulik, N., Ettrich, R., Kren, V., and Bezouska, K. (2011)
Enzymatic characterization and molecular modeling of an evolutio-
narily interesting fungal β-N-acetylhexosaminidase. FEBS J. 278,
4
C7G.
2
469−2484.
AUTHOR INFORMATION
(8) Jiang, Y. L., Yu, W. L., Zhang, J. W., Frolet, C., Guilmi, A. M. D.,
■
Zhou, C. Z., Vernet, T., and Chen, Y. (2011) Structural basis for the
substrate specificity of a novel β-N-acetylhexosaminidase StrH protein
from Streptococcus pneumoniae R6. J. Biol. Chem. 286, 43004−43012.
Corresponding Author
E-mail: nicolas.doucet@iaf.inrs.ca. Fax: (450) 686-5501. Tel.:
450) 687-5010, ext. 4212.
*
(
(9) Liu, T., Zhang, H., Liu, F., Chen, L., Shen, X., and Yang, Q.
Author Contributions
(2011) Active-pocket size differentiating insectile from bacterial
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
chitinolytic β-N-acetyl-D-hexosaminidases. Biochem. J. 438, 467−474.
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Funding
This work was supported by a Natural Sciences and
Engineering Research Council of Canada (NSERC) Discovery
grant (RGPIN 402623-2011), an NSERC Engage grant, and a
(11) Williams, S. J., Mark, B. L., Vocadlo, D. J., James, M. N. G., and
Withers, S. G. (2002) Aspartate 313 in the Streptomyces plicatus
hexosaminidase plays a critical role in substrate-assisted catalysis by
orienting the 2-acetamido group and stabilizing the transition state. J.
Biol. Chem. 277, 40055−40065.
“
́ ́
Fonds de Recherche Quebec−Sante” (FRQS) Research
Scholar Junior 1 Career Award (to N.D). N.N.T was the
recipient of a Ph.D. scholarship from the “Fondation
Universitaire Armand-Frappier de l′INRS”. W.A.O. is funded
by the United Kingdom Biotechnology and Biological Sciences
Research Council (Grant BB/K003836/1).
(12) Howard, M. B., Ekborg, N. A., Weiner, R. M., and Hutcheson, S.
W. (2003) Detection and characterization of chitinases and other
chitin-modifying enzymes. J. Ind. Microbiol. Biotechnol. 30, 627−635.
(
13) Chen, J. K., Shen, C. R., and Liu, C. L. (2010) N-
Acetylglucosamine: production and applications. Mar. Drugs 8,
Notes
2
493−2516.
The authors declare no competing financial interest.
(14) Sashiwa, H., Fujishima, S., Yamano, N., Kawasaki, N.,
Nakayama, A., Muraki, E., Hiraga, K., Oda, K., and Aiba, S. (2002)
Production of N-acetyl-D-glucosamine from α-chitin by crude enzymes
from Aeromonas hydrophila H2330. Carbohydr. Res. 337, 761−763.
ACKNOWLEDGMENTS
The authors thank Julie Payet (INRS) for technical assistance
■
(
15) Pichyangkura, R., Kudan, S., Kuttiyawang, K., Sukwattanasinitt,
and Franco
̧
is Lep
́
ine (INRS) for mass spectrometry. Data
M., and Aiba, S. (2002) Quantitative production of 2-acetoamodo-2-D-
glucose from crystalline chitin by bacterial Chitinase. Carbohydr. Res.
collection was performed at the Diamond Light Source
beamlines IO3 and IO4 (Oxfordshire, UK) and staff are
thanked for provision of data collection facilities.
3
37, 557−559.
(16) Saito, A., Fujii, T., Yoneyama, T., Redenbach, M., Ohno, T., and
Watanabe, T. (1999) High-multiplicity of Chitinase genes in
ABBREVIATIONS
Streptomyces coelicolor A3(2). Biosci. Biotechnol. Biochem. 63, 710−718.
■
(
17) Saito, A., Ishizaka, M., Francisco, P. B. J., Fujii, T., and
HEX, β-N-acetylhexosaminidase; ScHEX, β-N-acetylhexosami-
nidase from Streptomyces coelicolor A3(2); GlcNAc, N-
acetylglucosamine; GalNAc, N-acetylgalactosamine; GH, glyco-
side hydrolase; pNP, para-nitrophenyl
Miyashita, K. (2000) Transcriptional co-regulation of five Chitinase
genes scattered on the Streptomyces coelicolor A3(2) chromosome.
Microbiology 146, 2937−2946.
̃
́
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L., Thomson, N. R., James, K. D., Harris, D. E., Quail, M. A., Kieser,
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Huang, C. H., Kieser, T., Larke, L., Murphy, L., Oliver, K., O’Neil, S.,
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β-N-acetylhexosaminidase: a target for the design of antifungal drugs.
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