title: A novel member of the subtilisin-like protease family from Streptomyces albogriseolus.
abstract: We previously isolated three extracellular endogenous enzymes from a Streptomyces albogriseolus mutant strain which were targets of Streptomyces subtilisin inhibitor (SSI) (S. Taguchi, A. Odaka, Y. Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In the present study, of the three enzymes the largest one, with a molecular mass of 45 kDa (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), termed SAM-P45, has been characterized in detail. The entire gene encoding SAM-P45 was cloned as an approximately 10-kb fragment from S. albogriseolus S-3253 genomic DNA into an Escherichia coli host by using a shuttle plasmid vector. The amino acid sequence corresponding to the internal region of SAM-P45, deduced from the nucleotide sequence of the gene, revealed high homology, particularly in three regions around the active-site residues (Asp, His, and Ser), with the amino acid sequences of the mature domain of subtilisin-like serine proteases. In order to investigate the enzymatic properties of this protease, recombinant SAM-P45 was overproduced in Streptomyces coelicolor by using a strong SSI gene promoter. Sequence analysis of the SAM-P45 gene and peptide mapping of the purified SAM-P45 suggested that it is synthesized as a large precursor protein containing a large C-terminal prodomain (494 residues) in addition to an N-terminal preprodomain (23 and 172 residues). A high proportion of basic amino acids in the C-terminal prodomain was considered to serve an element interactive with the phospholipid bilayer existing in the C-terminal prodomain, as found in other membrane-anchoring proteases of gram-positive bacteria. It is noteworthy that SAM-P45 was found to prefer basic amino acids to aromatic or aliphatic amino acids in contrast to subtilisin BPN', which has a broad substrate specificity. The hydrolysis by SAM-P45 of the synthetic substrate (N-succinyl-L-Gly-L-Pro-L-Lys-p-nitroanilide) most preferred by this enzyme was inhibited by SSI, chymostatin, and EDTA. The proteolytic activity of SAM-P45 was stimulated by the divalent cations Ca2+ and Mg2+. From these findings, we conclude that SAM-P45 interacts with SSI and can be categorized as a novel member of the subtilisin-like serine protease family.
authors.affiliation: Department of Biological Science and Technology, Science University of Tokyo, Noda-shi, Chiba, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Suzuki
authors.forename: S
authors.initials: S
authors.lastname: Taguchi
authors.forename: S
authors.initials: S
authors.lastname: Yamada
authors.forename: S
authors.initials: S
authors.lastname: Kojima
authors.forename: K I
authors.initials: KI
authors.lastname: Miura
authors.forename: H
authors.initials: H
authors.lastname: Momose
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013381:Subtilisins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D014107:Tosyllysine Chloromethyl Ketone; D014108:Tosylphenylalanyl Chloromethyl Ketone; D013381:Subtilisins