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Published on: September 5, 2013
Genetic and biochemical characterization of glutamyl endopeptidase of Staphylococcus warneri M
K Yokoi1, M Kakikawa, H Kimoto
1Molecular Biology Group, Faculty of Engineering, Toyama University, 3190 Gofuku, Toyama 930-8555, Japan.
Abstract:
A Staphylococcus warneri strain M, newly isolated from processed seafood (smoked Watasenia scintillans), produced an extracellular protease. The protease, designated to as m-PROM (the mature form of PROM), selectively cleaved the carbonyl side of glutamic acid residues in beta-casein. Sequence of N-terminal 27 amino acids of m-PROM, RANVILPNNDRHQINDTTLGHYAPVTF, was found to be similar to those of other glutamyl endopeptidases, V8 protease (Staphylococcus aureus strain V8) and SPase (S. aureus ATCC 12600). To determine the complete primary structure and precursor of PROM, its gene (proM) was cloned and sequenced. The gene proM was found to encode for a protein of 316 amino acids. The amino acid residues from 64 to 90 completely coincided with the N-terminal 27 amino acids of the m-PROM, suggesting that the N-terminal 63 amino acids region of p-PROM (the precursor form of PROM) might be processed posttranslationally. Moreover, the whole amino acid sequence deduced from the primary structure of proM shows significant similarity to those of other glutamyl endopeptidases, V8 protease and SPase. These results suggested that PROM belongs to the glutamyl endopeptidase class. PROM, however, differs from V8 and SPase proteases in the processing site and the C-terminal region.
Insights
A novel extracellular protease, PROM, was isolated from Staphylococcus warneri. This glutamyl endopeptidase selectively cleaves beta-casein and shows unique processing sites compared to similar proteases.
Area of Science:
- Microbiology
- Enzymology
- Protein Chemistry
Background:
- Extracellular proteases play crucial roles in microbial physiology and interactions.
- Staphylococcus species are known producers of various proteases with different specificities.
- Characterizing novel proteases aids in understanding enzymatic mechanisms and potential applications.
Purpose of the Study:
- To isolate and characterize a novel extracellular protease from Staphylococcus warneri strain M.
- To determine the substrate specificity and primary structure of the protease, designated PROM.
- To elucidate the genetic basis and processing of PROM and compare it with known glutamyl endopeptidases.
Main Methods:
- Isolation and purification of extracellular protease from S. warneri strain M.
- Substrate cleavage analysis using beta-casein to determine specificity.
- N-terminal amino acid sequencing of the mature protease (m-PROM).
- Cloning and sequencing of the protease gene (proM) to determine the precursor structure.
- Bioinformatic analysis to compare PROM with other glutamyl endopeptidases.
Main Results:
- Staphylococcus warneri strain M produced an extracellular protease (PROM) that selectively cleaves beta-casein at glutamic acid residues.
- The N-terminal sequence of mature PROM (m-PROM) showed similarity to known glutamyl endopeptidases.
- Sequencing of the proM gene revealed a 316-amino acid precursor (p-PROM), with the N-terminal 63 amino acids likely undergoing post-translational processing.
- PROM shares significant sequence homology with V8 protease and SPase but differs in processing sites and C-terminal region.
Conclusions:
- PROM is identified as a novel glutamyl endopeptidase from Staphylococcus warneri.
- The enzyme exhibits specific cleavage activity on beta-casein, targeting glutamic acid residues.
- PROM represents a distinct member of the glutamyl endopeptidase family due to variations in its processing and C-terminal structure.
