Abstract
Bacterial extracellular peptidases are important for bacterial nutrition and organic nitrogen degradation in the ocean. While many peptidases of the M13 family from terrestrial animals and bacteria are studied, there has been no report on M13 peptidases from marine bacteria. Here, we characterized an M13 peptidase, PepS, from the deep-sea sedimentary strain Shewanella sp. E525-6, and investigated its substrate specificity and catalytic mechanism. The gene pepS cloned from strain E525-6 contains 2085 bp and encodes an M13 metallopeptidase. PepS was expressed in Escherichia coli and purified. Among the characterized M13 peptidases, PepS shares the highest sequence identity (47%) with Zmp1 from Mycobacterium tuberculosis, indicating that PepS is a new member of the M13 family. PepS had the highest activity at 30°C and pH 8.0. It retained 15% activity at 0°C. Its half life at 40°C was only 4 min. These properties indicate that PepS is a cold-adapted enzyme. The smallest substrate for PepS is pentapeptide, and it is probably unable to cleave peptides of more than 30 residues. PepS prefers to hydrolyze peptide bonds with P1′ hydrophobic residues. Structural and mutational analyses suggested that His531, His535 and Glu592 coordinate the catalytic zinc ion in PepS, Glu532 acts as a nucleophile, and His654 is probably involved in the transition state stabilization. Asp538 and Asp596 can stablize the orientations of His531 and His535, and Arg660 can stablize the orientation of Asp596. These results help in understanding marine bacterial peptidases and organic nitrogen degradation.
Highlights
In the ocean, High-Molecular-Weight Organic Nitrogen (HMW ON) produced by organisms in the seawater settles and accumulates in the sediment in the form of particulate organic nitrogen (PON) (Brunnegård et al, 2004)
In addition to proteases that are responsible for HMW ON degradation, bacteria secrete peptidases to degrade peptides into small oligopeptides and/or amino acids because bacteria generally prefer to absorb oligopeptides of 2–5 amino acid residues and cannot import oligopeptides of more than 35 amino acid residues
The results reveal the characteristics of the M13 peptidase PepS from a deep-sea sedimentary bacterium, 1http://merops.sanger.ac.uk/cgi-bin/famsum?family=M13 which sheds light on the degradation of peptides by deep-sea sedimentary bacteria
Summary
High-Molecular-Weight Organic Nitrogen (HMW ON) produced by organisms in the seawater settles and accumulates in the sediment in the form of particulate organic nitrogen (PON) (Brunnegård et al, 2004). CF6-2 (Zhao et al, 2012) and myroilysin from Myroides profundi D25 (Chen et al, 2009) These bacterial extracellular proteases can hydrolyze HMW ON, such as collagen and elastin, into Low-Molecular-Weight Dissolved Organic Nitrogen (LMW DON), such as peptides and amino acids, and may play important roles in sedimentary PON degradation (Zhao et al, 2008, 2012; Chen et al, 2009). Reports on peptidases from marine bacteria are rather few It is still unclear how peptides are degraded into utilizable oligopeptides and/or amino acids for bacterial nutrition in marine sediment. The results reveal the characteristics of the M13 peptidase PepS from a deep-sea sedimentary bacterium, 1http://merops.sanger.ac.uk/cgi-bin/famsum?family=M13 which sheds light on the degradation of peptides by deep-sea sedimentary bacteria
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