Purification, characterization cloning, and sequencing of metalloendopeptidase from Streptomyces septatus TH-2.
Streptomyces septatus TH-2 secretes a large amount of a protease when cultured on a medium containing K(2)HPO(4) and glucose. The enzyme was purified to homogeneity by a three-step procedure. This enzyme had a molecular mass of approximately 35kDa, and was particularly inhibited by EDTA and phosphoramidon. Its substrate specificity was investigated using novel fluorescence energy transfer combinatorial libraries. The protease was found to prefer Phe and Tyr at the P(1) position, a hydrophobic or basic residue at the P(2) position, and a basic or small residue at the P(3) position. Its gene was cloned and sequenced, and its deduced amino acid sequence contained an HEXXH consensus sequence for zinc binding, confirming that it encodes metalloendopeptidase. The primary structure of the enzyme showed 40 and 69% identities with that of thermolysin from Bacillus thermoproteolyticus and that of a metalloendopeptidase from Streptomyces griseus, respectively.
Hatanaka
Tadashi
T
Research Institute for Biological Sciences (RIBS), Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan. hatanaka@bio-ribs.com
Yoshiko Uesugi
Jiro Arima
JA
Iwabuchi
Masaki
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001407:Bacillus / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D003470:Culture Media / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D005947:Glucose / Q000378:metabolism; D006020:Glycopeptides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013054:Spectrophotometry, Atomic; D013302:Streptomyces / Q000201:enzymology*; D013305:Streptomyces griseus / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature; D013820:Thermolysin / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D003470:Culture Media; D006020:Glycopeptides; D014443:Tyrosine; D010649:Phenylalanine; D004492:Edetic Acid; D008666:Metalloendopeptidases; D013820:Thermolysin; D005947:Glucose; D015032:Zinc; C008890:phosphoramidon