PMID:24459146

Citation	Gadwal, S, Korotkov, KV, Delarosa, JR, Hol, WG and Sandkvist, M (2014) Functional and structural characterization of Vibrio cholerae extracellular serine protease B, VesB. J. Biol. Chem. 289:8288-98
Abstract	The chymotrypsin subfamily A of serine proteases consists primarily of eukaryotic proteases, including only a few proteases of bacterial origin. VesB, a newly identified serine protease that is secreted by the type II secretion system in Vibrio cholerae, belongs to this subfamily. VesB is likely produced as a zymogen because sequence alignment with trypsinogen identified a putative cleavage site for activation and a catalytic triad, His-Asp-Ser. Using synthetic peptides, VesB efficiently cleaved a trypsin substrate, but not chymotrypsin and elastase substrates. The reversible serine protease inhibitor, benzamidine, inhibited VesB and served as an immobilized ligand for VesB affinity purification, further indicating its relationship with trypsin-like enzymes. Consistent with this family of serine proteases, N-terminal sequencing implied that the propeptide is removed in the secreted form of VesB. Separate mutagenesis of the activation site and catalytic serine rendered VesB inactive, confirming the importance of these features for activity, but not for secretion. Similar to trypsin but, in contrast to thrombin and other coagulation factors, Na(+) did not stimulate the activity of VesB, despite containing the Tyr(250) signature. The crystal structure of catalytically inactive pro-VesB revealed that the protease domain is structurally similar to trypsinogen. The C-terminal domain of VesB was found to adopt an immunoglobulin (Ig)-fold that is structurally homologous to Ig-folds of other extracellular Vibrio proteins. Possible roles of the Ig-fold domain in stability, substrate specificity, cell surface association, and type II secretion of VesB, the first bacterial multidomain trypsin-like protease with known structure, are discussed.
Links	PubMed PMC3961656 Online version:10.1074/jbc.M113.525261
Keywords	Amino Acid Sequence; Cholera/microbiology; Enzyme Activation; Humans; Models, Molecular; Molecular Sequence Data; Protein Structure, Tertiary; Sequence Alignment; Serine Proteases/chemistry; Serine Proteases/metabolism; Sodium/metabolism; Structural Homology, Protein; Thrombin/chemistry; Trypsin/chemistry; Vibrio cholerae/chemistry; Vibrio cholerae/enzymology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
VIBCH:Q9KSQ6	enables	GO:0008236: serine-type peptidase activity	ECO:0000315: mutant phenotype evidence used in manual assertion		F		Seeded From UniProt	complete
VIBCH:Q9KSQ6		GO:0005615: extracellular space	ECO:0000314:		C		Supernatant and cell extracts from three different bacteria culture strains(VesA,VesB,VesC) were subject to SDS-Page and Western blot using anti-VesB antibodies to demonstrate secretion. Figure 4b	complete CACAO 10713
VIBCH:Q9KSQ6		GO:0008236: serine-type peptidase activity	ECO:0000315:		F		When subjected to serine protease inhibitors, its protease activity was effected.Figure 2 Protease activity was measured using different commercially available synthetic peptides to determine substrate specificity and subjected to a serine peptidase specific inhibitor. Figure 3A and 3B	complete CACAO 10716

References

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