TAILIEUCHUNG - Báo cáo khoa học: Role of active-site residues of dispersin B, a biofilm-releasing b-hexosaminidase from a periodontal pathogen, in substrate hydrolysis

Dispersin B (DspB), a family 20b-hexosaminidase from the oral pathogen Aggregatibacter actinomycetemcomitans, cleaves b(1,6)-linked N-acetylglu-cosamine polymer. In order to understand the substrate specificity of DspB, we have undertaken to characterize several conserved and noncon-served residues in the vicinity of the active site. | ỊFEBS Journal Role of active-site residues of dispersin B a biofilm-releasing b-hexosaminidase from a periodontal pathogen in substrate hydrolysis Suba G. A. Manuel Chandran Ragunath Hameetha B. R. Sait Era A. Izano Jeffrey B. Kaplan and Narayanan Ramasubbu Department of OralBiology University of Medicine and Dentistry of New Jersey Newark NJ USA Keywords biofilm detachment dispersin B enzyme mechanism hydrolysis site-directed mutagenesis Correspondence N. Ramasubbu Department of OralBiology C-634 MSB UMDNJ 185 South Orange Ave Newark NJ 07103 USA Fax 1 973 972 0045 Tel 1 973 972 0704 E-mail ramasun1@ Received 28 August 2007 revised 30 September 2007 accepted 1 October 2007 doi Dispersin B DspB a family 20 b-hexosaminidase from the oral pathogen Aggregatibacter actinomycetemcomitans cleaves b 1 6 -linked N-acetylglu-cosamine polymer. In order to understand the substrate specificity of DspB we have undertaken to characterize several conserved and nonconserved residues in the vicinity of the active site. The active sites of DspB and other family 20 hexosaminidases possess three highly conserved acidic residues several aromatic residues and an arginine at subsite -1. These residues were mutated using site-directed mutagenesis and characterized for their enzyme activity. Our results show that a highly conserved acid pair in b-hexosaminidases D183 and E184 and E332 play a critical role in the hydrolysis of the substrates. pH activity profile analysis showed a shift to a higher pH in the optimal activity for the E184Q mutant suggesting that this residue might act as the acid base catalyst. The reduction in kcat observed for Y187A and Y278A mutants suggests that the Y187 residue unique to DspB located on a loop might play a role in substrate specificity and be a part of subsite 1 whereas the hydrogen-bond interaction between Y278A and the N-acetyl group might help to stabilize the transition state. Mutation of W237 and W330 .

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