From the Departments of Biochemistry and Molecular
Biology and
Human Genetics, University of
Manitoba, Winnipeg, Manitoba, R3E 0W3, Canada, the ¶ Department of
Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7,
Canada, and the ** Center for Cancer Research, Massachusetts Institute
of Technology, Cambridge, Massachusetts 02139
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have sequenced the Streptomyces
plicatus -N-acetylhexosaminidase
(SpHex) gene and identified the encoded protein as a member
of family 20 glycosyl hydrolases. This family includes human
-N-acetylhexosaminidases whose deficiency results in
various forms of GM2 gangliosidosis. Based upon the x-ray
structure of Serratia marcescens chitobiase
(SmChb), we generated a three-dimensional model of
SpHex by comparative molecular modeling. The overall structure of the enzyme is very similar to homology modeling-derived structures of human
-N-acetylhexosaminidases, with
differences being confined mainly to loop regions. From previous
studies of the human enzymes, sequence alignments of family 20 enzymes,
and analysis of the SmChb x-ray structure, we selected and
mutated putative SpHex active site residues.
Arg162
His mutation increased Km
40-fold and reduced Vmax 5-fold, providing the
first biochemical evidence for this conserved Arg residue
(Arg178 in human
-N-acetylhexosaminidase A
(HexA) and Arg349 in SmChb) as a
substrate-binding residue in a family 20 enzyme, a finding consistent
with our three-dimensional model of SpHex. Glu314
Gln reduced Vmax
296-fold, reduced Km 7-fold, and altered the pH
profile, consistent with it being the catalytic acid residue as
suggested by our model and other studies. Asp246
Asn
reduced Vmax 2-fold and increased
Km only 1.2-fold, suggesting that
Asp246 may play a lesser role in the catalytic mechanism of
this enzyme. Taken together with the x-ray structure of
SmChb, these studies suggest a common catalytic mechanism
for family 20 glycosyl hydrolases.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Streptomyces plicatus -N-acetylhexosaminidase
(SpHex)1 is a
glycosyl hydrolase that removes N-acetylglucosamine or
N-acetylgalactosamine residues from the nonreducing end of
oligosaccharides and their conjugates. This enzyme may be important for
the efficient degradation of polysaccharides by Streptomyces
species (1). In humans, the importance of
-N-acetylhexosaminidase is illustrated by the fatal
neurodegenerative disorders that result from its deficiency.
Human -N-acetylhexosaminidase A (HexA), a heterodimer of
subunits
(encoded by HEXA) and
(encoded by
HEXB), is essential for the degradation of ganglioside
GM2 (2). Mutations in HEXA or HEXB
cause Tay-Sachs and Sandhoff disease, respectively. Structure/function studies of HexA have been limited by the difficulty in producing levels
of recombinant HexA in mammalian expression systems that are sufficient
for kinetic analysis (3, 4).
The classification of glycosyl hydrolases into families based on amino acid sequence similarity has greatly facilitated the identification of evolutionary and mechanistic relationships between these enzymes (5-7). To date, 63 glycosyl hydrolase families have been identified, 24 of which have a three-dimensional structure determined for at least one member.
Family 20 contains -N-acetylhexosaminidases and
chitobiases (EC 3.2.1.52) and are thought to use an acid/base mechanism that involves a proton donor and a nucleophile (8). Serratia marcescens chitobiase (SmChb) is the only family 20 member for which a three-dimensional structure has been determined (9). According to the 1.9-Å resolution crystallographic model,
SmChb appears to lack the nucleophilic residue necessary for
catalysis. Alternatively, Tews et al. (9) suggest that the
N-acetyl group on the nonreducing
N-acetylglucosamine residue of the chitobiose substrate may
act as the nucleophile (substrate-assisted catalysis) (9). The
structure also predicts that Glu540 is the proton donor and
that Arg349 is directly involved in substrate binding. Both
residues are conserved among family 20 enzymes. In human HexA,
mutagenesis studies of the
- and
-subunit residues homologous to
SmChb Glu540,
-Glu323 and
-Glu355, respectively, have been identified as likely
proton donors (10, 11). Fernandes et al. (10) suggested that
-Asp258, previously predicted to be a proton donor in
HexA, fulfills a lesser role in the catalytic mechanism of this enzyme.
These studies on HexA provide biochemical support for
Glu540 of SmChb as the proton donor in family 20 enzymes; however, there is no biochemical evidence in the literature
that supports the suggestion that Arg349 of
SmChb or its homologue in other family 20 enzymes is a
substrate binding residue.
We have sequenced the gene encoding SpHex, classified it as
a new member of family 20 glycosyl hydrolases, and constructed a
three-dimensional model of the SpHex enzyme by comparative
molecular modeling. In conjunction with the molecular modeling,
site-directed mutagenesis was used to demonstrate a role for
Arg162 (human HexA, -Arg178;
SmChb, Arg349) in substrate binding. Our
biochemical analysis of Glu314 (human HexA,
-Glu323; SmChb, Glu540) and
Asp246 (human HexA,
-Asp258;
SmChb, Asp448) mutations further substantiate
the role of these residues in family 20 enzymes. These studies indicate
that the catalytic mechanism of these enzymes may be conserved.
Finally, multiple sequence alignments of the enzymes indicate that
SpHex is significantly more related in structure to human
HexA and -B than SmChb.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recombinant DNA Methods and Strains-- Escherichia coli strain JM109 was used for all plasmid manipulations and fusion protein production. Plasmid DNA was purified using the Nucleobond AX purification system (Macherey-Nagel). Restriction enzymes and Klenow fragment were from New England Biolabs. Restriction enzyme digests and filling-in 3'-recessed ends of DNA were according to New England Biolabs instructions. T4 DNA ligase was from Boehringer Mannheim, and DNA ligations were performed according to their instructions. Liquid cultures of S. plicatus (American Type Culture Collection number 27800) were grown at 25 °C in International Streptomyces Project tryptone yeast extract broth (ISP medium 1) (Difco). Stocks of S. plicatus were maintained on ISP agar plates at 4 °C.
Sequencing the SpHex Clone--
Clone 36, expressing
SpHex (1), was previously identified by screening a -ZAP
library using 4-methylumbelliferyl (4-MU) glycosides of
N-acetylglucosamine oligosaccharides as a substrate for an
in plate assay (12). Clone 36 was found to have a 5.0-kb insert and
expressed a 60-kDa fusion protein (1). Based on this, the coding region
for SpHex was predicted to be within a 1.8-kb region of the
insert adjacent to the
-galactosidase gene in the vector. The 1.8-kb
region, bounded by EcoRI (5') and SmaI (3')
sites, was restriction enzyme-mapped and subcloned into pBluescript (pBS+) (Stratagene) as two smaller fragments. The subclones
were sequenced at the National Centers of Excellence core sequencing
facility (Toronto, Canada) by the dideoxy chain termination method
(13). Sequencing was done on double-stranded DNA and covered both
strands. To clarify the sequence in some areas, we used
sequence-specific primers to sequence single-stranded DNA produced from
a 1.8-kb EcoRI/SmaI clone (psHEX-1.8).
Comparative Molecular Modeling-- Atomic coordinates for SmChb were obtained from the Brookhaven National Laboratory Protein Data Bank (Protein Data Bank code 1qbb) and used as a template for molecular modeling. The best alignment between the amino acid sequences of SpHex, SmChb, and the 13 other known family 20 glycosyl hydrolases was obtained using ClustalW1.7. To improve the alignment, sequences that increased the frequency of insertions and deletions were sequentially removed, and the remaining sequences were realigned. This minimized the number of insertions and deletions within the SpHex sequence and maximized alignments of the secondary structural elements as visualized using the graphics program O (19). The final sequence alignment between SpHex and SmChb was optimized manually by comparing initial models of SpHex to the x-ray structure of SmChb using O (20).
Using Homology (Insight software, BioSym Technologies, Inc.), the aligned amino acid sequence of SpHex was substituted onto the SmChb backbone atomic coordinates. Initial side chain steric clashes were relieved using a rotamer library containing the most favorable residue side chain conformations. No amino acid insertions into the SpHex model were required, and gaps were manually joined using the program O. Serious steric clashes were relieved manually. The model was placed in a primitive lattice (P1 space group) and subjected to energy minimization using a conjugate gradient target function implemented in X-PLOR (21, 22). The cell parameters were set sufficiently large to avoid intermolecular contacts during minimization. Minimization was performed with the van der Waals and electrostatic terms turned on. The model was minimized until convergence was reached, as judged by the root mean square of the energy gradient (average derivative < 0.1 kcal/mol/Å). The overall quality of the model was assessed with the program PROCHECK (23), using comparison values typical for a 2.0-Å resolution x-ray structure.Construction of the pMAL-c2-SpHex Fusion Protein Vector--
A
construct pMAL-c2-SpHex (a gift from New England Biolabs)
was made by subcloning the insert from clone 36 (BamHI/Hind
III) into pMAL-c2 (XmnI/HindIII). The
BamHI site was filled in to facilitate subcloning and to
allow for correct translation of the maltose-binding protein
(MBP)-SpHex fusion
protein.2 To remove 3.2 kb of
extraneous DNA, pMAL-c2-SpHex was digested with
SmaI and HindIII, the HindIII site was
blunt-ended, and both the larger vector (7 kb) and a 1.8-kb
(SmaI/SmaI) fragment containing the coding
region, were gel-purified and ligated to generate pmHEX-1.8. A plasmid
with the fragment ligated in the correct orientation was chosen using
4-methylumbelliferyl--N-acetylglucosaminide (4-MUG)
(Toronto Research Chemicals Inc.) as substrate for an in plate assay
(12).
Site-directed Mutagenesis--
Single-stranded DNA from
psHEX-1.8 was isolated (24) using M13K07 (New England Biolabs) and
mutations were introduced following instructions from Bio-Rad based on
the procedure of Kunkel et al. (25) but using T7 polymerase
(26). Phosphorylated oligonucleotides (ACGT Corp., Toronto, Canada)
5'-TCGGCGGCGACCAGGCGCACTCCAC-3', 5'-CACGAAGTCGTAGGTGACGT-3', and
5'-GTCCCCGAGATCAACATGCCGGGCC-3' were used to create the substitutions
G1208C (Glu314 Gln), G753A and G754T
(Arg162
His), and G1004A (Asp246
Asn),
respectively. Newly synthesized double-stranded DNA isolated and
purified using the Geneclean II kit (4). The product (20-40%) was
transformed into E. coli and colonies containing mutant
plasmid constructs were identified by polymerase chain reaction
amplification of the relevant region followed by restriction enzyme
digestion (base change G1208C created a ScrFI site, G753A
and G754T combined destroyed a MspI site, and G1004A
destroyed a TaqI site). Fragments containing the mutation(s)
were subcloned into pmHEX-1.8 (AscI/BsrGI fragment contained either G753A and G754T or G1004A;
BsrGI/AgeI fragment contained G1208C) to create
the various mutant pmHEX-1.8(s). The subcloned regions were sequenced
to confirm that only the desired base changes had occurred.
Purification of SpHex-MBP Fusion and Cleavage with Factor Xa Protease-- SpHex-MBP fusion protein was prepared in batches based on instructions from New England Biolabs except that fusion protein expression was induced at 25 °C, and E. coli suspensions were supplemented with 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 2 µg/ml pepstatin A prior to lysis. Incubation of the purified fusion protein with 2% (w/w) factor Xa protease (New England Biolabs) in 20 mM Tris-Cl, pH 7.4, 200 mM NaCl, 1 mM EDTA, pH 7.4, containing 2 mM CaCl2 for 18-24 h at room temperature facilitated cleavage of the MBP from SpHex. The fusion protein and cleavage products were visualized using SDS-PAGE as described elsewhere (27).
Protein and Enzyme Assays--
Protein concentrations were
determined by the Bradford method (28) using -globulin as the
standard. To determine the pH optima for mutant and wild type
SpHex, activities for each were measured using 4-MUG as a
substrate in 10 mM Na2HPO4, 6 mM citric acid, 0.3% bovine serum albumin ranging from pH
2.0 to 9.0. After a 45-min incubation at 25 °C, each reaction was
quenched with 0.1 M glycine-carbonate buffer, pH 10. The
fluorescent product, 4-MU, was measured (excitation, 364 nm; emission,
448 nm) using a Hitatchi F-2000 fluorescence spectrophotometer. Kinetic
data for mutant and wild type SpHex were obtained in an
identical manner at the pH optimum of the wild type enzyme using 4-MUG
concentrations ranging from 0.033 to 5 mM.
Km and Vmax values were determined using
the direct linear plot method (29).
Generation of Antibodies against SpHex-MBP Fusion Protein-- Rabbit anti-SpHex-MBP fusion protein antibodies were raised at the National Biological Laboratories (Oakbank, Canada). Briefly, purified SpHex-MBP fusion protein emulsified with Freund's complete adjuvant was injected subcutaneously at multiple sites. Two weeks later, the antigen was emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple sites. This step was repeated 3 weeks later. Serum was collected 1 week after the final injection.
Western Blot Analysis--
To obtain a crude S. plicatus cytosolic fraction for immunoblotting experiments, the
cells were collected by centrifugation at 5000 × g for
10 min and then resuspended in 4 ml of TNE (20 mM Tris-Cl,
pH 7.4, 200 mM NaCl, 1 mM EDTA, pH 7.4)
containing 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml
aprotinin, 5 µg/ml leupeptin, and 2 µg/ml pepstatin A. The
suspension was homogenized with a Dounce homogenizer for 1 min at
4 °C and sonicated (6 times for 15 s) at 100 watts on ice using
a Braun-sonic 1510 (B. Braun Melsungen AG). The lysate was centrifuged
at 20000 × g for 20 min, and the supernatant,
considered to be the crude cytosolic fraction, was stored at
20 °C.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
DNA Sequence Analysis of SpHex--
Originally, clone 36 was
classified as a -N-acetylhexosaminidase on the basis of
its substrate specificity (1). In a search for enzymes related to human
-N-acetylhexosaminidase that might be useful for
structure/function analysis, we determined the nucleotide sequence and
deduced amino acid sequence of the enzyme (SpHex) encoded by
clone 36. This sequence did not include an initiating Met codon,
indicating that a portion of the 5'-end of the gene was missing. This
was not surprising, given that SpHex had to be produced as a
-galactosidase fusion to allow its expression in E. coli
(1). However, Western blot analysis demonstrated that the molecular
masses of the recombinant and native SpHex were
indistinguishable (Fig. 1,
lanes 3 and 4), suggesting that only a small portion of the
5'-end of the SpHex gene was missing from the clone. An
additional clone (ps5HEX-1.0) containing the 5'-end of the gene was
obtained, and 284 bp of new sequence was determined. The complete
coding sequence revealed a 1683-bp open reading frame, encoding 561 amino acids.
|
|
Comparative Molecular Modeling--
A pairwise alignment between
the SmChb and SpHex using MaxHom revealed a
sequence identity of 30%. When the alignment was restricted to
SmChb domain 3, an /
-barrel structure that contains the enzyme active site on the C-terminal end, the sequence identity was
45%. Domain 3 is the largest of four SmChb domains.
Alignment of SpHex residues
Pro149-Pro498 with domain 3 encompassed 350 residues (63%) of the total amino acid sequence of
SpHex.
|
Analysis of the SpHex Model--
The final energy-minimized model
of SpHex is shown as a ribbon diagram
in Fig. 3, A and B.
PROCHECK analysis before and after energy minimization demonstrated
that energy minimization dramatically improved the overall geometry and
relaxed most of the bad contacts within the model. Of the 10 bad
contacts remaining, none occur within the core of the model; only one
contact, between the last two residues in the model, is less than 2.5 Å (Leu497 C Pro498 N, 1.3 Å). A
Ramachandran map indicated that 94.3% of the residues had
,
angles within core and allowed regions, 3.5% had
,
angles
within generously allowed regions, and 2.1% (6 residues) had
disallowed
,
angles. The residues with disallowed
,
angles (Val214, Leu255, Ala369,
Leu404, Ala433, Thr474) all reside
in loop structures on the outer surfaces of the model, primarily at
locations spliced together to compensate for deletions.
|
Enzyme Purification and Digestion with Factor Xa Protease-- The pmHEX-1.8 constructs of wild type and mutant SpHex produced high level fusion protein expression in E. coli, yielding approximately 0.5 mg/100 ml culture. The final protein concentrations for all purifications were brought to 1-2 mg/ml by eluting the fusion protein with 0.5 ml of elution/storage buffer. Purified wild type and mutant SpHex-MBP fusion protein was stable for at least 48 h at room temperature and at least 2 weeks at 4 °C.
The purified SpHex-MBP fusion protein was digested with factor Xa protease to assess the effects of the MBP on enzyme activity. There was no difference in the pH optimum or Km of undigested or digested enzyme (data not shown). Western blot analysis using the anti-SpHex-MBP antibody indicated that factor Xa cleaved the SpHex-MBP fusion protein into a 55-kDa protein predicted to be recombinant SpHex and the 43-kDa MBP (Fig. 1, lane 3). Anti-SpHex-MBP antibody also detected a 55-kDa protein in the cytosol of S. plicatus (Fig. 1, lane 4), indicating that the recombinant and native SpHex are of the same approximate molecular mass. Two higher molecular weight proteins were also detected in the S. plicatus cytosol; they are thought to be a result of nonspecific cross-reactivity of the antibody to other proteins in the fraction. Interestingly, SpHex was detected in the spent medium of S. plicatus cultures after approximately 5 days of growth, suggesting that S. plicatus may secrete this enzyme into the medium upon expression (Fig. 1, lane 5).Kinetic Analysis of Wild-type and Mutant SpHex--
Based upon
multiple sequence alignments of family 20 enzymes, previous mutagenesis
studies of human HexA (44-47) and the SmChb structure (9),
we created the following mutations in SpHex: Arg162 His, Asp246
Asn and
Glu314
Gln. Since the kinetics of the
SpHex-MBP fusion and factor Xa-digested fusion protein were
indistinguishable from each other when using 4-MUG as substrate,
kinetic analysis was restricted to the SpHex-MBP fusion
protein. The resulting kinetic analyses (Table
II) are consistent with our predicted
structure of SpHex (Fig. 3, C and D).
The model of SpHex predicts that the Arg162
His mutation would significantly affect the substrate binding capacity
of the enzyme (Fig. 3C). Indeed, this mutation increased the
Km 40-fold as compared with wild type
SpHex. The observed 5-fold decrease in the
Vmax observed for this mutation probably
reflects the inability of the enzyme to efficiently dock the substrate.
By virtue of conservation, not only does this mutation provide direct
biochemical evidence to support the predicted role of
Arg349 in SmChb, it is the first example of this
conserved Arg acting as a substrate-binding residue. This same mutation
is associated with the B1 variant form of Tay-Sachs disease in humans,
but previous studies of the mutation in the human enzyme showed that
the equivalent Arg residue in human HexA is not involved in substrate
binding (46, 47). The discrepancy may reflect the difficulties in obtaining accurate kinetic data from the human enzyme.
|
pH Profile Analysis--
The pH profiles of wild type and mutant
SpHex can be seen in Fig. 4.
The pH optimum of wild type SpHex was found to be 3.0. Although the pH optimum of this enzyme is quite broad, an environment more acidic than pH 3.0 resulted in a steady loss of activity. Interestingly, the Glu314 Gln mutation changed the pH
profile from that of a broad bell-shaped curve to more a hyperbolic
shaped pH curve with no distinctive peak in the pH profile. According
to our model and Tews et al. (9), Glu314 must be
protonated in order for catalysis to occur. This is reflected in the
acidic pH optimum seen for wild-type SpHex. Gln does not undergo ionization; hence, the loss of a distinct peak at pH 3.0 suggests that the ionization state of Glu314 is responsible
for the pH dependence seen for the wild-type enzyme.
|
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Peter C. Loewen for providing access and guidance to his computer facilities, to Sharon Wong-Madden of New England Biolabs for providing the initial pMAL-c2-SpHex fusion construct as well as guidance for optimal protein production, and to the Canadian Genetic Diseases Network sequencing core facility for generating the initial SpHex nucleotide sequence.
![]() |
FOOTNOTES |
---|
* This work was funded by the Medical Research Council (MRC) of Canada Grant MT-11708 (to B. T. R.) and by a grant from MRC of Canada to the Group in Protein Structure and Function (to M. N. G. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF063001.
§ Supported by a Manitoba Health Research Council studentship.
Present address: Dept. of Pathology and Laboratory Medicine,
University of Louisville School of Medicine, Louisville, KY 40292.
§§ Supported by a Medical Research Council of Canada scholarship. To whom correspondence should be addressed. Tel.: 204-789-3218; Fax: 204-789-3900; E-mail: traine{at}ms.umanitoba.ca.
1
The abbreviations used are:
SpHex, S. plicatus
-N-acetylhexosaminidase; HexA, and -B, human
-hexosaminidase A and B, respectively; GM2,
GalNAc
(1,4)-[N-acetylneuraminic acid
(2,3)-]-Gal
(1-4)-Glc-ceramide; SmChb, S. marcescens chitobiase; 4-MU, 4-methylumbelliferyl; 4-MUG, 4-methylumbelliferyl-
-N-acetylglucosaminide; MBP,
maltose-binding protein; ISP, international Streptomyces
project; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s);
kb, kilobase pair(s).
2 S. Wong-Madden, personal communication.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|