INTRODUCTION

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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 G_M2 (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

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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).

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. K_m and V_max 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

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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.

View larger version (60K): [in this window] [in a new window]   Fig. 1.   Comparison of recombinant and native SpHex. To directly compare molecular masses of recombinant and native SpHex, the MBP was removed from affinity-purified SpHex-MBP fusion with factor Xa protease as described under "Experimental Procedures." A crude S. plicatus cytosolic fraction was prepared after growth in ISP medium for 5 days ("Experimental Procedures"). Protein samples were resolved by 7.5% SDS-PAGE, transferred to nitrocellulose, and incubated with polyclonal anti-SpHex-MBP antibody as described under "Experimental Procedures." The antigen-antibody complex was visualized using the ECL protocol. Mobility of prestained protein standards (New England Biolabs) are indicated on the left. Lane 1, 10 ng of affinity-purified MBP-LacZ fusion (expressed from pMAL-c2 with no insert in the polylinker); lane 2, 10 ng of affinity-purified SpHex-MBP fusion protein; lane 3, 10 ng of incomplete factor Xa digest of SpHex-MBP fusion protein; lane 4, 15 µg of S. plicatus crude cytosolic fraction; lane 5, 4 µg of spent ISP medium used to grow the S. plicatus culture. IB, immunoblot.

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.

View larger version (62K): [in this window] [in a new window]   Fig. 2.   Sequence alignment SpHex and SmChb. The alignment was constructed using ClustalW1.7 (see "Experimental Procedures"). Amino acid numbering is indicated on the right. Asterisks indicate identical residues, whereas filled circles indicate similar residues. Arrows indicate residues subjected to mutational analysis (Arg162, Glu314, and Asp246). Secondary structures are indicated along the top of alignment (, -helix; , -sheet). Secondary structures responsible for forming the /-barrel structure are numbered (1-8 and 1-6, 8; note there is no 7 helix).

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.

View larger version (49K): [in this window] [in a new window]   Fig. 3.   SpHex model complexed with chitobiose. The model contains residues Pro149-Pro498. Panel A shows a ribbon diagram of the model looking down the axis of the /-barrel. Panel B is turned 90° on the y axis with respect to panel A and shows a side view of the barrel. The substrate complexes with loop structures on the C-terminal end of the /-barrel and secondary structures forming the /-barrel are indicated. Red cylinders, -helix; green ribbons, -sheets. Panel C shows residues predicted to be involved in the catalytic mechanism of the SpHex. Panel D depicts the predicted hydrophobic interactions occurring between the enzyme and substrate.

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.

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 K_m 40-fold as compared with wild type SpHex. The observed 5-fold decrease in the V_max 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.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Analysis of pH profiles for wild type and mutant SpHex. Catalytic activity for wild type and mutant SpHex was measured in 10 mM Na2HPO4, 6 mM citric acid, 0.3% bovine serum albumin ranging from pH 2.0 to 9.0 using 4-MUG as a substrate as described under "Experimental Procedures." After a 45-min incubation at 25 °C, each reaction was quenched with 0.1 M glycine-carbonate buffer, pH 10, and the fluorescent product, 4-MU, was measured by fluorometry. Activity is indicated as an average percentage of maximal activity (n = 3).