Extracellular metalloprotease gene of Streptomyces cacaoi: structure, nucleotide sequence and characterization of the cloned gene product.
The gene (npr) encoding an extracellular neutral metalloprotease (Npr) from Streptomyces cacaoi YM15 was cloned in Streptomyces lividans using pIJ702 as a vector. The nucleotide (nt) sequence of npr was determined. The deduced open reading frame encoded 550 amino acids (aa) (60 kDa) with a putative signal sequence of 34 aa at the N terminus. High-resolution S1 mapping identified the transcriptional start point at about 132-134 nt upstream from the start codon. The nt sequences at both -10 and -35 regions resemble the consensus sequence of typical Escherichia coli promoters and a fragment containing the promoter was functional in an E. coli promoter probe plasmid. In vitro transcription and translation of the cloned npr sequence revealed a 60-kDa protein product, correlated with the sequence data but not with the size (35 kDa) of the extracellular Npr. The N-terminal aa sequence in conjunction with the aa composition analyses on the purified mature Npr led to the conclusion that it was processed from the 60-kDa pre-proenzyme form encoded by npr. The Npr protease contained putative zinc ligand-binding regions and two repeated motifs, Asp-Ser-Gly, similar to the active site residues of the aspartic acid and retroviral proteases.
Chang
P C
PC
Institute of Biochemistry, National Yang-Ming Medical College, Taipei, Taiwan.
Kuo
T C
TC
Tsugita
A
A
Lee
Y H
YH
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D015342:DNA Probes; D004591:Electrophoresis, Polyacrylamide Gel; D008666:Metalloendopeptidases / Q000235:genetics*; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013302:Streptomyces / Q000235:genetics*; D014158:Transcription, Genetic
D015342:DNA Probes; D008666:Metalloendopeptidases