Auto- and Hetero-Catalytic Processing of the N-Terminal Propeptide Promotes the C-Terminal Fibronectin Type III Domain-Mediated Dimerization of a Thermostable Vpr-like Protease

Vpr-like proteases are widely distributed in bacteria and fungi and are involved in processing lantibiotics, degrading collagen, keratin, and fibrin, and pathogenesis of microbes. The dissection of the roles of individual domains in enzyme maturation and oligomerization is crucial for understanding the action mechanisms of these multidomain proteases. Microbial Vpr-like proteases are extracellular multidomain subtilases with diverse functions and can form oligomers, but their maturation and oligomerization mechanisms remain to be elucidated. Here, we report a novel Vpr-like protease (BTV) from thermophilic bacterium Brevibacillus sp. WF146. The BTV precursor comprises a signal peptide, an N-terminal propeptide, a subtilisin-like catalytic domain with an inserted protease-associated (PA) domain, two tandem fibronectin type III domains (Fn1 and Fn2), and a C-terminal propeptide. The BTV proform (pro-BTV) could be autoprocessed into the mature form (mBTV) via two intermediates lacking the N- or C-terminal propeptide, respectively, and the C-terminal propeptide delays the autocatalytic maturation of the enzyme. By comparison, pro-BTV is more efficiently processed into mBTV by protease TSS from strain WF146. Purified mBTV is a Ca2+-dependent thermostable protease, showing optimal activity at 60 degrees C and retaining more than 60% of activity after incubation at 60 degrees C for 8 h. The PA domain is important for enzyme stability and contributes to the substrate specificity of BTV by restricting the access of protein substrates to the active site. The proform and mature form of BTV exist as a monomer and a homodimer, respectively, and the dimerization is mediated by the Fn1 and Fn2 domains. The N-terminal propeptide of BTV not only acts as intramolecular chaperone and enzymatic inhibitor but also inhibits the homodimerization of the enzyme. The removal of the N-terminal propeptide leads to a structural adjustment of the enzyme and thus promotes enzyme dimerization. IMPORTANCE Vpr-like proteases are widely distributed in bacteria and fungi and are involved in processing lantibiotics, degrading collagen, keratin, and fibrin, and pathogenesis of microbes. The dissection of the roles of individual domains in enzyme maturation and oligomerization is crucial for understanding the action mechanisms of these multidomain proteases. Our results demonstrate that hetero-catalytic maturation of the extracellular Vpr-like protease BTV of Brevibacillus sp. WF146 is more efficient than autocatalytic maturation of the enzyme. Moreover, we found that the C-terminal tandem fibronectin type III domains rather than the PA domain mediate the dimerization of mature BTV, while the N-terminal propeptide inhibits the dimerization of the BTV proform. This study provides new insight into the activation and oligomerization mechanisms of Vpr-like proteases.