Structural Basis for the Substrate Specificity of a Novel β-N-Acetylhexosaminidase StrH Protein from Streptococcus pneumoniae R6

Abstract

The ␤-N-acetylhexosaminidase (EC 3.2.1.52) from glycoside hydrolase family 20 (GH20) catalyzes the hydrolysis of the ␤-N-acetylglucosamine (NAG) group from the nonreducing end of various glycoconjugates. The putative surface-exposed N-acetylhexosaminidase StrH/Spr0057 from Streptococcus pneumoniae R6 was proved to contribute to the virulence by removal of ␤(1,2)-linked NAG on host defense molecules following the cleavage of sialic acid and galactose by neuraminidase and ␤-galactosidase, respectively. StrH is the only reported GH20 enzyme that contains a tandem repeat of two 53% sequence-identical catalytic domains (designated as GH20-1 and GH20-2, respectively). Here, we present the 2.1 Å crystal structure of the N-terminal domain of StrH (residues Glu-175 to Lys-642) complexed with NAG. It adopts an overall structure similar to other GH20 enzymes: a (␤/␣) 8 TIM barrel with the active site residing at the center of the ␤-barrel convex side. The kinetic investigation using 4-nitrophenyl N-acetyl-␤-D-glucosaminide as the substrate demonstrated that GH20-1 had an enzymatic activity (k cat /K m ) of one-fourth compared with GH20-2. The lower activity of GH20-1 could be attributed to the substitution of active site Cys-469 of GH20-1 to the counterpart Tyr-903 of GH20-2. A complex model of NAG␤(1,2)Man at the active site of GH20-1 combined with activity assays of the corresponding site-directed mutants characterized two key residues Trp-443 and Tyr-482 at subsite ؉1 of GH20-1 (Trp-876 and Tyr-914 of GH20-2) that might determine the ␤(1,2) substrate specificity. Taken together, these findings shed light on the mechanism of catalytic specificity toward the ␤(1,2)-linked ␤-N-acetylglucosides.

Crystal Structures of a Glycoside Hydrolase Family 20 Lacto-N-biosidase from Bifidobacterium bifidum

“…The N-domain has an ␣/␤ topology with a seven-stranded ␤-sheet exposed to the surface, and two ␣-helices buried in the interface with the subsequent barrel domain. The N-domain and the barrel domain correspond to the two conserved domains of typical GH20 ␤-HexNAcases (21,24,25,(27)(28)(29)(30)(31)(32). The N-domain and the barrel domain of BbLNBase are structurally similar to the domains I and II, respectively, of ␤-HexNAcase from Streptomyces plicatus (SpHex) (Fig.…”

Structural Insights into the Substrate Specificity of a 6-Phospho-β-glucosidase BglA-2 from Streptococcus pneumoniae TIGR4

“…Glucose and G6P standards were quantified by HPLC analysis using various concentrations ranging from 0.1 to 1 mM. The mixing buffer containing 20% acetonitrile and 100 mM Na 2 HPO 4 /NaH 2 PO 4 , pH 7.0 was processed as described previously (38) for equilibration of the column (Eclipse XDB-C18 column, 4.6 ϫ 150 mm; Agilent), and separation of the components was effected at a flow rate of 1 ml/min. Retention times of monosaccharides were determined by comparison with standard solutions.…”

Cell Surface Glycoside Hydrolases of Streptococcus gordonii Promote Growth in Saliva

“…Four putative cell wall-anchored GHs were identified from S. gordonii DL1, of which three were predicted to act on host glycans based on their homology with pneumococcal BgaA (13,14), StrH (15,16), and EndoD (17). In the present study, we introduced unmarked, in-frame deletions in the corresponding genes and verified cell surface expression of the encoded proteins on the resulting mutant strains by their reactions with anti-BgaA, anti-StrH, or anti-EndoD antisera (Fig.…”

“…N-terminally polyhistidine (6ϫHis)-tagged recombinant proteins containing the putative catalytic region of each cell surface GH were prepared for use as immunogens. The nucleotide sequences for amino acid residues 80 to 609 of BgaA (13,14), 136 to 507 of StrH (15,16), and 113 to 762 of EndoD (17), including in-frame 5=-BamHI and 3=-HindIII cloning sites and the requisite start (ATG) and stop codons (TAA or TGA), were optimized for expression in E. coli, synthesized, and cloned into pTrcHisB (Invitrogen) by Blue Heron Biotechnology, Inc. Plasmid-bearing E. coli EC100 bacteria were grown at 37°C with aeration in Luria-Bertani broth supplemented with ampicillin (150 g/ml). At an A 600 of 0.4, the cultures were rapidly chilled in ice water, and isopropyl thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM.…”