Expression and characterization of the biofilm‐related and carnosine‐hydrolyzing aminoacylhistidine dipeptidase from Vibrio alginolyticus

Abstract

Vibrio alginolyticus is one of twelve recognized marine Vibrio species that have been identified as pathogenic for humans and marine animals. This species causes infection in shrimps, fish, shellfish and squids, as well as in humans who are infected via consumption of undercooked seafood or exposure of wounds to warm seawater in coastal areas [1][2][3]. V. alginolyticus infects grouper culture by forming a biofilm in the intestine and causes fish mortality due to gastroenteritis syndrome [4]. A disease outbreak of shrimp farming in 1996 was also attributed to V. alginolyticus virulence [5]. In infected humans, clinical symptoms include gastroenteritis, wound infections and septicemia [6][7][8] and, more rarely, ear infections, chronic diarrhea exclusively in AIDS patients, conjunctivitis and post-traumatic intracranial infection [9][10][11]. Thus, prevention, early 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 HisHis 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 HisAETag-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.Abbreviations CPG2, Pseudomonas sp. carboxypeptidase G2; hAcy1, human aminoacylase-1; MH, metallopeptidase H clan; OPA, O-phthaldialdehyde; PepD, aminoacylhistidine dipeptidase.

Crystal Structure and Mutational Analysis of Aminoacylhistidine Dipeptidase from Vibrio alginolyticus Reveal a New Architecture of M20 Metallopeptidases

“…Biochemical studies of V. alginolyticus PepD revealed its metal-dependent characteristics (36) and optimal activation of Secondary structure elements are shown in red for ␣-helices and blue for ␤-strands. Gray spheres represent the zinc ions.…”

“…apo-PepD has been observed with various divalent metal ions, including Mn 2ϩ , Co 2ϩ , Ni 2ϩ , Cu 2ϩ , and Cd 2ϩ (36). Previous studies have shown that the addition of Co 2ϩ ions to apo-PepD increases the enzymatic activity by a factor of ϳ1.…”

“…The V. alginolyticus PepD has a broader specificity for Xaa-His dipeptides and is sensitive to inhibition by bestatine and L-3,4-dihydroxyphenylalanine (36,37). Bioinformatic analysis of the V. alginolyticus PepD protein revealed high sequence homology to that from other Vibrio species (94 -76% identity) and bacteria (75-63%).…”

“…We have previously cloned and sequenced a biofilm-related and carnosine-hydrolyzing pepD gene from Vibrio alginolyticus (36,37). The V. alginolyticus PepD has a broader specificity for Xaa-His dipeptides and is sensitive to inhibition by bestatine and L-3,4-dihydroxyphenylalanine (36,37).…”

“…Nevertheless, sequence analysis revealed that putative active site residues for catalysis are conserved in PepD and related di-zinc enzymes in the M20 family (6,23 . Asp 173 was present in homologs with aminopeptidase/dipeptidase specificity, although members of aminoacylase/carboxypeptidase contained a glutamic acid in the same position (36).…”