Molecular characterization of fervidolysin, a subtilisin-like serine protease from the thermophilic bacterium Fervidobacterium pennivorans

Abstract

The fls gene encoding fervidolysin, a keratin-degrading proteolytic enzyme from the thermophilic bacterium Fervidobacterium pennivorans, was isolated using degenerate primers combined with Southern hybridization and inverse polymerase chain reaction. Further sequence characterization demonstrated that the 2.1-kb fls gene encoded a 699-amino-acid preproenzyme showing high homology with the subtilisin family of the serine proteases. It was cloned into a pET9d vector, without its signal sequence, and expressed in Escherichia coli. The heterologously produced fervidolysin was purified by heat incubation followed by ion exchange chromatography and emerged in the soluble fraction as three distinct protein bands, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal-sequence analysis of these bands and their comparison with that determined from biochemically purified keratinase and its predicted protein sequence, identified them as a 73-kDa fervidolysin precursor, a 58-kDa mature fervidolysin, and a 14-kDa fervidolysin propeptide. Using site-directed mutagenesis, the active-site histidine residue at position 79 was replaced by an alanine residue. The resulting fervidolysin showed a single protein band corresponding in size to the 73-kDa fervidolysin precursor, indicating that its proteolytic cleavage resulted from an autoproteolytic process. Knowledge-based modeling experiments showed a distinctive binding region for subtilases, in which binding of the propeptide could take place prior to autoproteolysis. Assays using keratin and other proteinaceous substrates did not display fervidolysin activity, perhaps because of the tight binding of the propeptide in the substrate-binding site, where it could then function as an inhibitor.

SufA – a novel subtilisin-like serine proteinase of Finegoldia magna

“…However, none of the tested methods were successful, and the protein remained proteolytically inactive. This has been reported also for other recombinantly expressed subtilases (Harris et al, 2003;Kluskens et al, 2002).…”

Intracellular Localization and Trafficking of Serine Proteinase AhSub and Cysteine Proteinase AhCP of Acanthamoeba healyi

“…vealed that the pre domain of AhSub and the prepro domain of AhCP are essential for appropriate intracellular localization and trafficking of these proteinases. Subtilisins are usually synthesized as prepro enzymes, which are later posttranslationally activated to the active enzymes by cleavage of the pre-and propeptides (11). The roles of preand propeptides of subtilisin trafficking in bacteria have been studied by biochemical and molecular biological investigations.…”

Cloning and Expression of Islandisin, a New Thermostable Subtilisin from Fervidobacterium islandicum , in Escherichia coli

“…However, SDS-PAGE analysis revealed that the prosequence is not cleaved off by autoprocessing and remains attached to active islandisin. The recombinant fervidolysin, on the other hand, did show autoprocessing but failed to become active, perhaps due to tight attachment of the propeptide (18). In a second purification step, the recombinant enzyme was purified by hydroxyapatite column chromatography.…”

“…A protease-encoding gene from Fervidobacterium pennivorans was detected, amplified by PCR and cloned in E. coli. The recombinant protein, however, did not show any activity (18).…”