標題: Expression and characterization of the biofilm-related and carnosine-hydrolyzing aminoacylhistidine dipeptidase from Vibrio alginolyticus
作者: Wang, Ting-Yi
Chen, Yi-Chin
Kao, Liang-Wei
Chang, Chin-Yuan
Wang, Yu-Kuo
Liu, Yen-Hsi
Feng, Jen-Min
Wu, Tung-Kung
生物科技學系
Department of Biological Science and Technology
關鍵字: aminoacylhistidine dipeptidase;biofilm;carnosinase;metallopeptidase H clan;Vibrio alginolyticus
公開日期: 1-Oct-2008
摘要: The biofilm-related and carnosine-hydrolyzing aminoacylhistidine dipeptidase (pepD) gene from Vibrio alginolyticus was cloned and sequenced. The recombinant PepD protein was produced and biochemically characterized and the putative active-site residues responsible for metal binding and catalysis were identified. The recombinant enzyme, which was identified as a homodimeric dipeptidase in solution, exhibited broad substrate specificity for Xaa-His and His-Xaa dipeptides, with the highest activity for the His-His dipeptide. Sequence and structural homologies suggest that the enzyme is a member of the metal-dependent metallopeptidase family. Indeed, the purified enzyme contains two zinc ions per monomer. Reconstitution of His.Tag-cleaved native apo-PepD with various metal ions indicated that enzymatic activity could be optimally restored when Zn(2+) was replaced with other divalent metal ions, including Mn(2+), Co(2+), Ni(2+), Cu(2+) and Cd(2+), and partially restored when Zn(2+) was replaced with Mg(2+). Structural homology modeling of PepD also revealed a 'catalytic domain' and a 'lid domain' similar to those of the Lactobacillus delbrueckii PepV protein. Mutational analysis of the putative active-site residues supported the involvement of His80, Asp119, Glu150, Asp173 and His461 in metal binding and Asp82 and Glu149 in catalysis. In addition, individual substitution of Glu149 and Glu150 with aspartic acid resulted in the partial retention of enzymatic activity, indicating a functional role for these residues on the catalysis and zinc ions, respectively. These effects may be necessary either for the activation of the catalytic water molecule or for the stabilization of the substrate-enzyme tetrahedral intermediate. Taken together, these results may facilitate the design of PepD inhibitors for application in antimicrobial treatment and antibody-directed enzyme prodrug therapy.
URI: http://dx.doi.org/10.1111/j.1742-4658.2008.06635.x
http://hdl.handle.net/11536/8306
ISSN: 1742-464X
DOI: 10.1111/j.1742-4658.2008.06635.x
期刊: FEBS JOURNAL
Volume: 275
Issue: 20
起始頁: 5007
結束頁: 5020
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