微生物胞外枯草杆菌酶是通常用于商业应用的高活性蛋白水解酶。这些枯草杆菌酶以其无活性原形合成,在前肽结构域的控制下成熟为活性蛋白酶。在嗜温细菌原草蛋白酶中,前肽的功能是作为强制性伴侣和枯草杆菌酶催化结构域的抑制剂。相比之下,嗜高温古细菌原草酶的前肽主要作为紧密抑制剂,对于枯草酶的折叠不是必需的。尚不清楚嗜热前肽的这种更强的抑制活性是否会导致它们对同源枯草杆菌酶的选择性更高。与混杂的嗜温前肽相反。这里,我们证明了Pernisine的前肽,一种超热稳定的古细菌枯草杆菌酶,强烈地与Pernisine相互作用并抑制Pernisine,但不是同源的枯草杆菌蛋白酶Carlsberg和蛋白酶K。相反,Pernisine前肽容易被枯草杆菌蛋白酶Carlsberg和蛋白酶K降解。未加工性质的催化域也容易降解,但在性质自动加工成非活性性质后变得蛋白水解稳定,非共价复合前肽:pernisine。这允许在嗜温温度下通过枯草杆菌蛋白酶Carlsberg和蛋白酶K降解Pernisine前肽来有效地激活自动加工的复合前肽:Pernisine。此外,我们证明了活性pernisine分子被未加工的propernisine催化降解后释放的前肽抑制。这突出了嗜高温前肽对其同源枯草杆菌酶的高抑制效力及其在调节枯草杆菌酶成熟中的重要性,以防止未加工的枯草杆菌酶前体被过早地活化的分子降解。重要性许多微生物将蛋白酶分泌到它们的环境中以降解蛋白质底物用于它们的生长。这些胞外酶的重要组是枯草杆菌酶,在实际应用中也得到了广泛的应用。这些枯草杆菌酶被它们的前肽域抑制,在原草蛋白酶成熟过程中降解。这里,我们证明了Pernisine的前肽,一种来自超嗜热古细菌的朊病毒降解枯草杆菌酶,强烈抑制具有非常高的结合亲和力的pernisine。这种互动被证明是高度选择性的,由于嗜温的Pernisine前肽被嗜温的同源物迅速降解。这反过来又允许在较低温度下通过嗜温枯草杆菌酶快速反式激活属性,这可能会简化用于商业用途的活性食品的制备程序。这项研究中报道的结果表明,嗜高温枯草杆菌酶前肽进化成为相关的枯草杆菌酶成熟的紧密和选择性调节剂,以防止其在高温下过早激活。
Microbial extracellular subtilases are highly active proteolytic enzymes commonly used in commercial applications. These subtilases are synthesized in their inactive proform, which matures into the active protease under the control of the
propeptide domain. In mesophilic bacterial prosubtilases, the
propeptide functions as both an obligatory chaperone and an inhibitor of the subtilase catalytic domain. In contrast, the propeptides of hyperthermophilic archaeal prosubtilases act mainly as tight inhibitors and are not essential for subtilase folding. It is unclear whether this stronger inhibitory activity of hyperthermophilic propeptides results in their higher selectivity toward their cognate subtilases, in contrast to promiscuous mesophilic propeptides. Here, we showed that the propeptide of pernisine, a hyperthermostable archaeal subtilase, strongly interacts with and inhibits pernisine, but not the homologous subtilisin Carlsberg and proteinase K. Instead, the pernisine
propeptide was readily degraded by subtilisin Carlsberg and proteinase K. In addition, the catalytic domain of unprocessed propernisine was also susceptible to degradation but became proteolytically stable after autoprocessing of propernisine into the inactive, noncovalent complex propeptide:pernisine. This allowed efficient transactivation of the autoprocessed complex propeptide:pernisine through degradation of pernisine propeptide by subtilisin Carlsberg and proteinase K at mesophilic temperature. Moreover, we demonstrated that active pernisine molecules are inhibited by the propeptide that is released after pernisine-catalyzed degradation of the unprocessed propernisine catalytic domain. This highlights the high inhibitory potency of the hyperthermophilic propeptide toward its cognate subtilase and its importance in regulating subtilase maturation, to prevent the degradation of the unprocessed subtilase precursors by the prematurely activated molecules. IMPORTANCE Many microorganisms secrete proteases into their environment to degrade protein substrates for their growth. The important group of these extracellular enzymes are subtilases, which are also widely used in practical applications. These subtilases are inhibited by their
propeptide domain, which is degraded during the prosubtilase maturation process. Here, we showed that the propeptide of pernisine, a prion-degrading subtilase from the hyperthermophilic archaeon, strongly inhibits pernisine with extraordinarily high binding affinity. This interaction proved to be highly selective, as pernisine propeptide was rapidly degraded by mesophilic pernisine homologs. This in turn allowed rapid transactivation of propernisine by mesophilic subtilases at lower temperatures, which might simplify the procedures for preparation of active pernisine for commercial use. The results reported in this study suggest that the hyperthermophilic subtilase
propeptide evolved to function as tight and selective regulator of maturation of the associated prosubtilase to prevent its premature activation under high temperatures.