Cloning, expression and characterization of a 46.5-kDa metallopeptidase from Bacillus halodurans H4 sharing properties with the pitrilysin family.
A 1242 base pair DNA fragment from Bacillus halodurans H4 isolated from alkaline sediments of Lake Bogoria (Kenya) coding for a potential protease was cloned and sequenced. The hexa-histidine-tagged enzyme was overexpressed in Escherichia coli and was purified in one step by immobilized-metal affinity chromatography (IMAC) on Ni-NTA resin. The protease (ppBH4) presents an inverted zincin motif, HXXEH, which defines the inverzincin family. It shares several biochemical and molecular properties with the clan ME family M16 metallopeptidases (pitrilysins), as well as with database hypothetical proteins that are potential M16 family enzymes. Thus, like insulysin and nardilysin, but contrary to bacterial pitrilysin, ppBH4 is inactivated by sulfhydryl alkylating agents. On the other hand, like bacterial pitrilysin, ppBH4 is sensitive to reducing agents. The enzymatic activity of ppBH4 is limited to substrates smaller than proteins. In contrast to insulin, dynorphin and insulin B-chain are very good substrates for ppBH4 and several cleavage sites are common with those observed with well-characterized pitrilysins. As deduced from amino acid sequence, as well as determined by gel-filtration and SDS-polyacrylamide gel electrophoresis, ppBH4 is an active monomer of 46.5 kDa. This feature distinguishes ppBH4 from all other enzymes of the pitrilysin family so far described whose molecular masses range from 100 to 140 kDa.
Dabonné
Soumaila
S
Unité de Biotechnologie, Biocatalyse et Biorégulation, CNRS-UMR 6204, Laboratoire de Biochimie, F44322 Nantes Cedex 3, France.
Moallic
Claire
C
Sine
Jean-Pierre
JP
Niamké
Sébastien
S
Dion
Michel
M
Colas
Bernard
B
D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification* / Q000201:enzymology*; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics; D045726:Metalloproteases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D045726:Metalloproteases; D008666:Metalloendopeptidases; C046583:pitrilysin