Evidence for Propeptide‐Assisted Folding of the Calcium‐Dependent Protease of the Cyanobacterium Anabaena

Abstract

The Ca"-dependent protease of the cyanobacterium Anabuena vuriabilis is a cytoplasmic enzyme with a substrate specificity like trypsin. Its previously published DNA sequence [Maldener, I., Lockau,Genet. 225, 113-1201 contained a sequencing error. Here we report the corrected sequence which shows, that the Ca2+-protease belongs to the family of subtilases (subtilisin-like serine proteases). Consistent with its cytoplasmic localization, a pre-sequence is not found.The enzyme is produced as a precursor with a large amino-terminal propeptide. Expression of the pro-region and mature region (protease domain) in Escherichia coli cells in trans demonstrates that formation of the active enzyme requires the propeptide. The results demonstrate that propeptide-assisted protein folding also occurs with cytoplasmic enzymes, in support of the hypothesis that this mechanism is a widespread phenomenon.

HreP, an In Vivo - Expressed Protease of Yersinia enterocolitica , Is a New Member of the Family of Subtilisin/Kexin-Like Proteases

“…Although HreP shows the highest similarity to PrcA of A. variabilis, PrcA is autocatalytically processed after an Arg residue, in contrast to processing after Lys by HreP. Furthermore, the PrcA cleavage site does not appear to have a consensus sequence (R-E-F-R-Q-R*) typical for subtilisin/kexinlike proteases (5). This suggests that HreP and PrcA cleave different substrates and may also have different functions.…”

“…Only one prokaryotic protease, PrcA of A. variabilis, showed significant similarity. Both HreP and PrcA share typically conserved residues that have been identified by sequence alignments and by a comparison of the crystal structures of several subtilases (5,32,46). One of three residues that are specific for the kexin subfamily of subtilases is conserved in HreP as well as in PrcA.…”

“…Three of four different hre mutant strains tested showed reduced virulence in the mouse model of infection, indicating their importance for pathogenesis. One of these, hreP (previously referred to as hre- 22), showed similarity to the protease PrcA of Anabaena variabilis (5,37,58). The importance of HreP for the pathogenesis of Y. enterocolitica is clearly reflected in the reduction of virulence of an hreP mutant strain by both a 50% lethal dose and an in vivo survival assay (58).…”

Ca 2+ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

“…The mature domains alone are not folded into an active form but are folded into an inactive form with molten-globular structure in the absence of propeptides (15,44). The requirement for a propeptide for maturation of its cognate mature domain has also been reported, not only for other members of the subtilase family (5,7,34), but also for other proteases (33,38,39,49,51,62). However, it remains to be determined whether archaeal subtilisins mature in a similar manner.…”

A calcium-stimulated serine peptidase from a true-branching cyanobacterium, Westiellopsis ramosa sp. nov.

“…Moreover, a calcium-dependent trypsin-like peptidase, a calcium-independent peptidase and a prolyl endopeptidase extracted from the cultures of Anabaena variabilis ATCC 29413 (A. variabilis 29413) have been shown to carry out proteolytic degradation of a number of natural and synthetic peptidase substrates (Lockau et al 1988;Strohmeier et al 1994;Maldener et al 1991), which suggests that extensive proteolytic activity inside the cells of heterocytous cyanobacteria does occur. A calcium-dependent peptidase from A. variabilis 29413 (Lockau et al 1988) which was also found in Anabaena 7120 was later characterized to be a subtilisin-like serine peptidase, PrcA, Ava_4009 (Baier et al 1996). With the advent of a full genome sequence of Anabaena sp.…”