Cloning and characterization of a metalloprotease from Vibrio harveyi strain AP6.
A metalloprotease gene pap6 was cloned from Vibrio harveyi strain AP6. Sequence analysis showed that pap6 was 2034 bp in length and predicted to encode a peptide of 677 amino acids with a molecular mass of 75 kDa. SDS-PAGE analysis of the purified Pap6 revealed that it was 35 kDa in size. N-terminal amino acid sequencing established that the mature protein began at Leu-191, suggesting that the preprotein of Pap6 was processed to generate a mature protease. Purified Pap6 was characterized as a zinc metalloprotease as it was inhibited by zinc- and metal-specific inhibitors such as 1, 10-phenanthroline, EGTA and EDTA. The deduced amino acid sequence revealed the presence of a zinc-binding motif HEXXH approximately 19aa approximately E. Substitution of these active site residues by site-directed mutagenesis caused significant losses in enzyme activity, thus demonstrating their involvement in catalysis. Pap6 was shown to digest a range of host proteins, including gelatin, fibronectin, and type IV collagen, indicating a potential role in pathogenesis.
Teo
Jeanette W P
JW
Programme in Environmental Microbiology, Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore.
Zhang
Lian Hui
LH
Poh
Chit Laa
CL
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010802:Phylogeny; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D014733:Vibrio / Q000201:enzymology / Q000235:genetics*; D015032:Zinc / Q000378:metabolism
D004269:DNA, Bacterial; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; D015032:Zinc