Cloning and sequence analysis of a novel metalloprotease gene from Vibrio parahaemolyticus 04.
The metalloprotease gene (vppC) from Vibrio parahaemolyticus 04 has been cloned and sequenced. The vppC gene contains an open reading frame of 2442 nucleotides encoding a polypeptide of 814 amino acids with a calculated molecular mass of 89,833 Da. The predicted amino acid sequence of VppC containing a zinc metalloprotease HEXXH consensus motif displays extensive homology to the collagenase from Vibrio alginolyticus. The activity of the recombinant protease produced in Escherichia coli was examined by gelatin zymography and proteolytic activity assays. The substrate specificity study showed that the type I collagen and synthetic collagenase substrate carbobenzoxy-glycyl-L-prolyl-glycyl-glycyl-L-prolyl-L-alanine were the best substrates, indicating that the cloned metalloprotease is indeed a collagenase. Multiple alignment analysis of the amino acid sequences and the enzymatic properties such as molecular mass and substrate specificity revealed three distinct classes of Vibrio metalloproteases. The identification of a new metalloprotease gene expands the role of Vibrio metalloproteases as a virulence factor for host infection.
Kim
Soo Kwang
SK
Division of Biological Sciences, College of Natural Sciences, Pusan National University, Pusan 609-735, South Korea.
Yang
Ji Young
JY
Cha
Jaeho
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D024042:Collagen Type I / Q000378:metabolism; D017364:Collagenases / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D008666:Metalloendopeptidases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014736:Vibrio parahaemolyticus / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D024042:Collagen Type I; D004269:DNA, Bacterial; D017364:Collagenases; D008666:Metalloendopeptidases