Active Subtilisin-Like Protease from a Hyperthermophilic Archaeon in a Form with a Putative Prosequence

Abstract

The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding, T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly ؊82 to Gly 316 ), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDSpolyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T. kodakaraensis subtilisin exists in a monomeric form. T. kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-AlaPro-Phe-p-nitroanilide only in the presence of the Ca 2؉ ion with an optimal pH and temperature of pH 9.5 and 80°C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80°C, 20 min at 90°C, and 7 min at 100°C.

Biochemical Properties of a Putative Signal Peptide Peptidase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

“…The complete genome sequence of T. kodakaraensis has recently been determined and annotated (13). As expected from the growth characteristics of this strain, the genome sequence revealed the presence of a large number of extracellular enzymes, including chitinase (36), ␣-amylase (35), and subtilisin-like protease (19). An orthologue search also revealed that T. kodakaraensis harbors a set of factors involved in protein secretion equivalent to those found in various hyperthermophilic archaea (see the Kyoto Encyclopedia of Genes and Genomes; http://www .genome.jp/kegg/).…”

Proteolytic inventory of Thermococcus kodakaraensis

“…They help the cells to utilize the proteins in extracellular media as source of essential amino acids (Hoaki et al, 1993). Two subtilisin like serine proteases (Tk-subtilisin and Tk-SP) and a thiol protease have been characterized and they have wide substrate specificity and are highly tolerant to temperature and pH (Kannan et al, 2001;Hirata et al, 2013;Morikawa et al, 1994). Leu→Pro mutation in Tk-subtilisin accelerated the maturation of Pro-Tk-subtilisin by reducing the binding ability of Tk-propeptide to Tk-subtilisin (Uehara et al, 2013).…”

“…There are three subtilisin-like serine protease precursors present in the genome of T. kodakaraensis (Table 4) and two of them, Tk-1675 and Tk-1689 have been characterized (Kannan et al, 2001;Rasool et al, 2010). Subtilisin like serine protease from T. kodakaraensis exhibit low similarity with other characterized subtilisins isolated from +2 ions are required for maturation of protease, in which it provides conformational change to the enzyme to autolyze its own prosequence.…”

Purification and Characterization of an Intracellular Chymotrypsin-Like Serine Protease fromThermoplasma volcanium

“…volcanium SP requires Ca 2þ ion for activity as other microbial SPs. 13,[31][32][33] Ions chelated by subtilases at either high or low affinity binding sites might stabilize enzymes by reducing flexibility, acting as salt or ion bridges. 34) The activity of Tp.…”

“…Most of these enzymes, which are recognized as the serine type, have been detected in the genera Pyrococcus (Pyrolysin and PfpI), 5,6) Desulforococcus (Archaelysin), 7) Sulfolobus, [8][9][10] and Thermococcus. [11][12][13] Genome sequence data revealed even more expansive proteolytic genotypes of these organisms than can be inferred from biochemical analyses. 14) A majority of these organisms are capable of growth on proteinaceous substrates.…”