A metalloprotease (MprIII) involved in the chitinolytic system of a marine bacterium, Alteromonas sp. strain O-7.
Alteromonas sp. strain O-7 secretes several proteins in addition to chitinolytic enzymes in response to chitin induction. In this paper, we report that one of these proteins, designated MprIII, is a metalloprotease involved in the chitin degradation system of the strain. The gene encoding MprIII was cloned in Escherichia coli. The open reading frame of mprIII encoded a protein of 1,225 amino acids with a calculated molecular mass of 137,016 Da. Analysis of the deduced amino acid sequence of MprIII revealed that the enzyme consisted of four domains: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The C-terminal extension (PkdDf) was characterized by four polycystic kidney disease domains and two domains of unknown function. Western and real-time quantitative PCR analyses demonstrated that mprIII was induced in the presence of insoluble polysaccharides, such as chitin and cellulose. Native MprIII was purified to homogeneity from the culture supernatant of Alteromonas sp. strain O-7 and characterized. The molecular mass of mature MprIII was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 115 kDa. The optimum pH and temperature of MprIII were 7.5 and 50 degrees C, respectively, when gelatin was used as a substrate. Pretreatment of native chitin with MprIII significantly promoted chitinase activity. Furthermore, the combination of MprIII and a novel chitin-binding protease (AprIV) remarkably promoted the chitin hydrolysis efficiency of chitinase.
Miyamoto
Katsushiro
K
Department of Microbiology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan.
Nukui
Eiji
E
Hirose
Mariko
M
Nagai
Fumi
F
Sato
Takaji
T
Inamori
Yoshihiko
Y
Tsujibo
Hiroshi
H
D020591:Alteromonas / Q000201:enzymology* / Q000378:metabolism; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002686:Chitin / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D004790:Enzyme Induction; D015870:Gene Expression; D008386:Marine Biology; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D014871:Water Microbiology
D004269:DNA, Bacterial; D002686:Chitin; D008666:Metalloendopeptidases