Ca2+-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

Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca²⁺ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca²⁺ at 80°C. This maturation process was completed within 30 min at 80°C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin*) but forms an inactive complex with T. kodakaraensis mat-subtilisin*, at lower temperatures. At 80°C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin*, and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca²⁺ but was activated upon incubation with Ca²⁺ at 80°C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin*, indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only ~5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin* and noncompetitively inhibited its activity with a K_i of 25 ± 3.0 nM at 20°C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.     

Folding pathway mediated by an intramolecular chaperone: dissecting conformational changes coincident with autoprocessing and the role of Ca(2+) in subtilisin maturation.

Subtilisin is produced as a precursor that requires its N-terminal propeptide to chaperone the folding of its protease domain. Once folded, subtilisin adopts a remarkably stable conformation, which has been attributed to a high affinity Ca(2+) binding site. We investigated the role of the metal ligand in the maturation of pro-subtilisin, a process that involves folding, autoprocessing and partial degradation. Our results establish that although Ca(2+) ions can stabilize the protease domain, the folding and autoprocessing of pro-subtilisin take place independent of Ca(2+) ion. We demonstrate that the stabilizing effect of calcium is observed only after the completion of autoprocessing and that the metal ion appears to be responsible for shifting the folding equilibrium towards the native conformation in both mature subtilisin and the autoprocessed propeptide:subtilisin complex. Furthermore, the addition of active subtilisin to unautoprocessed pro-subtilisin in trans does not facilitate precursor maturation, but rather promotes rapid autodegradation. The primary cleavage site that initiates this autodegradation is at Gln19 in the N-terminus of mature subtilisin. This corresponds to the loop that links alpha-helix-2 and beta-strand-1 in mature subtilisin and has indirect effects on the formation of the Ca(2+) binding site. Our results show that the N-terminus of mature subtilisin undergoes rearrangement subsequent to propeptide autoprocessing. Since this structural change enhances the proteolytic stability of the precursor, our results suggest that the autoprocessing reaction must be completed before the release of active subtilisin in order to maximize folding efficiency.     

Active subtilisin-like protease from a hyperthermophilic archaeon in a form with a putative prosequence

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 SDS-polyacrylamide 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-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca(2+) ion with an optimal pH and temperature of pH 9.5 and 80 degrees 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 degrees C, 20 min at 90 degrees C, and 7 min at 100 degrees C.     

An alternative mature form of subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis identified in the presence of Ca2+

Pro-Tk-SP from Thermococcus kodakaraensis consists of the four domains: N-propeptide, subtilisin (EC 3.4.21.62) domain, β-jelly roll domain and C-propeptide. To analyze the maturation process of this protein, the Pro-Tk-SP derivative with the mutation of the active-site serine residue to Cys (Pro-Tk-S359C), Pro-Tk-S359C derivatives lacking the N-propeptide (ProC-Tk-S359C) and both propeptides (Tk-S359C), and a His-tagged form of the isolated C-propeptide (ProC*) were constructed. Pro-Tk-S359C was purified mostly in an autoprocessed form in which the N-propeptide is autoprocessed but the isolated N-propeptide (ProN) forms a stable complex with ProC-Tk-S359C, indicating that the N-propeptide is autoprocessed first. The subsequent maturation process was analyzed using ProC-Tk-S359C, instead of the ProN:ProC-Tk-S359C complex. The C-propeptide was autoprocessed and degraded when ProC-Tk-S359C was incubated at 80 °C in the absence of Ca(2+). However, it was not autoprocessed in the presence of Ca(2+). Comparison of the susceptibility of ProC* to proteolytic degradation in the presence and absence of Ca(2+) suggests that the C-propeptide becomes highly resistant to proteolytic degradation in the presence of Ca(2+). We propose that Pro-Tk-SP derivative lacking N-propeptide (Val114-Gly640) represents a mature form of Pro-Tk-SP in a natural environment. The enzymatic activity of ProC-Tk-S359C was higher than (but comparable to) that of Tk-S359C, suggesting that the C-propeptide is not important for activity. However, the T(m) value of ProC-Tk-S359C determined by far-UV CD spectroscopy was higher than that of Tk-S359C by 25.9 °C in the absence of Ca(2+) and 7.5 °C in the presence of Ca(2+), indicating that the C-propeptide contributes to the stabilization of ProC-Tk-S359C.     

Accelerated refolding of subtilisin BPN' by tertiary-structure-forming mutants of its propeptide

The propeptide of subtilisin BPN', which functions as an intramolecular chaperone and a temporary inhibitor of subtilisin, is unique in that it acquires its three-dimensional structure by formation of a complex with the cognate protease. We previously showed that the successive amino acid replacements Ala47-->Phe, Gly13-->Ile, and Val65-->Ile in the propeptide to increase its hydrophobicity resulted in formation of a tertiary structure, accompanied by increased ability to bind to the protease and increased resistance to proteolysis. In this study, we examined the effects of these tertiary-structure-forming mutations on the intramolecular chaperone activity of the propeptide. The successive amino acid replacements mentioned above were introduced into pro-subtilisin*, possessing a Ser221-->Ala mutation in the catalytic residue. Refolding experiments were started by rapid dilution of the denatured pro-subtilisin*, and formation of tertiary structure in subtilisin was monitored kinetically by increase in tryptophan fluorescence. The wild-type pro-subtilisin* was found to refold with a rate constant of 4.8 x 10(-3) s(-1) in the equation describing an intramolecular process. The Ala47-->Phe replacement in the propeptide resulted in a 1.2-fold increase in the rate constant of subtilisin refolding. When the additional replacement Gly13-->Ile was introduced, refolding of subtilisin was substantially accelerated, and its kinetics could be fitted to a double exponential process composed of a fast phase with a rate constant of 2.1 x 10(-2) s(-1) and a slow phase with a rate constant of 4.5 x 10(-3) s(-1). The rate constant of the fast phase was increased slightly by a further replacement, Val65-->Ile. Since the slow phase is considered to correspond to proline isomerization, we concluded that tertiary-structure-forming mutations in the propeptide produce positive effects on its intramolecular chaperone activity through acceleration of the propeptide-induced formation of the tertiary structure of subtilisin BPN'.     

Crystal structure of unautoprocessed precursor of subtilisin from a hyperthermophilic archaeon: evidence for Ca2+-induced folding

The crystal structure of an active site mutant of pro-Tk-subtilisin (pro-S324A) from the hyperthermophilic archaeon Thermococcus kodakaraensis was determined at 2.3 A resolution. The overall structure of this protein is similar to those of bacterial subtilisin-propeptide complexes, except that the peptide bond linking the propeptide and mature domain contacts with the active site, and the mature domain contains six Ca2+ binding sites. The Ca-1 site is conserved in bacterial subtilisins but is formed prior to autoprocessing, unlike the corresponding sites of bacterial subtilisins. All other Ca2+-binding sites are unique in the pro-S324A structure and are located at the surface loops. Four of them apparently contribute to the stability of the central alphabetaalpha substructure of the mature domain. The CD spectra, 1-anilino-8-naphthalenesulfonic acid fluorescence spectra, and sensitivities to chymotryptic digestion of this protein indicate that the conformation of pro-S324A is changed from an unstable molten globule-like structure to a stable native one upon Ca2+ binding. Another active site mutant, pro-S324C, was shown to be autoprocessed to form a propeptide-mature domain complex in the presence of Ca2+. The CD spectra of this protein indicate that the structure of pro-S324C is changed upon Ca2+ binding like pro-S324A but is not seriously changed upon subsequent autoprocessing. These results suggest that the maturation process of Tk-subtilisin is different from that of bacterial subtilisins in terms of the requirement of Ca2+ for folding of the mature domain and completion of the folding process prior to autoprocessing.     

Increase in activation rate of Pro‐Tk‐subtilisin by a single nonpolar‐to‐polar amino acid substitution at the hydrophobic core of the propeptide domain

Tk‐subtilisin (Gly70‐Gly398) is a subtilisin homolog from Thermococcus kodakarensis. Active Tk‐subtilisin is produced from its inactive precursor, Pro‐Tk‐subtilisin (Gly1‐Gly398), by autoprocessing and degradation of the propeptide (Tk‐propeptide, Gly1‐Leu69). This activation process is extremely slow at moderate temperatures owing to high stability of Tk‐propeptide. Tk‐propeptide is stabilized by the hydrophobic core. To examine whether a single nonpolar‐to‐polar amino acid substitution at this core affects the activation rate of Pro‐Tk‐subtilisin, the Pro‐Tk‐subtilisin derivative with the Phe17→His mutation (Pro‐F17H), Tk‐propeptide derivative with the same mutation (F17H‐propeptide), and two active‐site mutants of Pro‐F17H (Pro‐F17H/S324A and Pro‐F17H/S324C) were constructed. The crystal structure of Pro‐F17H/S324A was nearly identical to that of Pro‐S324A, indicating that the mutation does not affect the structure of Pro‐Tk‐subtilisin. The refolding rate of Pro‐F17H/S324A and autoprocessing rate of Pro‐F17H/S324C were also nearly identical to those of their parent proteins (Pro‐S324A and Pro‐S324C). However, the activation rate of Pro‐F17H greatly increased when compared with that of Pro‐Tk‐subtilisin, such that Pro‐F17H is efficiently activated even at 40°C. The far‐UV circular dichroism spectrum of F17H‐propeptide did not exhibit a broad trough at 205–230 nm, which is observed in the spectrum of Tk‐propeptide. F17H‐propeptide is more susceptible to chymotryptic degradation than Tk‐propeptide. These results suggest that F17H‐propeptide is unfolded in an isolated form and is therefore rapidly degraded by Tk‐subtilisin. Thus, destabilization of the hydrophobic core of Tk‐propeptide by a nonpolar‐to‐polar amino acid substitution is an effective way to increase the activation rate of Pro‐Tk‐subtilisin.     

Autotomic behavior of the propeptide in propeptide-mediated folding of prosubtilisin E

The 77 residue propeptide at the N-terminal end of subtilisin E plays an essential role in subtilisin folding as a tailor-made intramolecular chaperone. Upon completion of folding, the propeptide is autoprocessed and removed by subtilisin digestion. This propeptide-mediated protein folding has been used as a paradigm for the study of protein folding. Here, we show by three independent methods, that the propeptide domain and the subtilisin domain show distinctive intrinsic stability that is obligatory for efficient autoprocessing of the propeptide domain. Two tryptophan residues, Trp106 and Trp113, on the surface of subtilisin located on one of the two helices that form the interface between the propeptide and the subtilisin domains play a key role in maintaining the distinctive instability of the propeptide domain, after completion of folding. When either of the Trp residues was substituted with Tyr, the characteristic biphasic heat denaturation profile of two domains unfolding was not observed, resulting in a single transition of denaturation. The results provide evidence that the propeptide not only plays an essential role in subtilisin folding, but upon completion of folding it behaves as an independent domain. Once the propeptide-mediated folding is completed, the propeptide domain is readily eliminated without interference from the subtilisin domain. This "autotomic" behavior of the propeptide may be a prevailing principle in propeptide-mediated protein folding.     

Four new crystal structures of Tk-subtilisin in unautoprocessed, autoprocessed and mature forms: insight into structural changes during maturation

Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-subtilisin) is matured from Pro-Tk-subtilisin upon autoprocessing and degradation of the propeptide. The crystal structures of the autoprocessed and mature forms of Tk-subtilisin were determined at 1.89 A and 1.70 A resolution, respectively. Comparison of these structures with that of unautoprocessed Pro-Tk-subtilisin indicates that the structure of Tk-subtilisin is not seriously changed during maturation. However, one unique Ca(2+)-binding site (Ca-7) is identified in these structures. In addition, the N-terminal region of the mature domain (Gly70-Pro82), which binds tightly to the main body in the unautoprocessed form, is disordered and mostly truncated in the autoprocessed and mature forms, respectively. Interestingly, this site is formed also in the unautoprocessed form when its crystals are soaked with 10 mM CaCl(2), as revealed by the 1.87 A structure. Along with the formation of this site, the N-terminal region (Leu75-Thr80) is disordered, with the scissile peptide bond contacting with the active site. These results indicate that the calcium ion binds weakly to the Ca-7 site in the unautoprocessed form, but is trapped upon autoprocessing. We propose that the Ca-7 site is required to promote the autoprocessing reaction by stabilizing the autoprocessed form, in which the new N terminus of the mature domain is structurally disordered. Furthermore, the crystal structure of the Tk-propeptide:S324A-subtilisin complex, which was formed by the addition of separately expressed proteins, was determined at 1.65 A resolution. This structure is virtually identical with that of the autoprocessed form, indicating that the interaction between the two domains is highly intensive and specific.     

Pro-subtilisin E: purification and characterization of its autoprocessing to active subtilisin E in vitro

The formation of active subtilisin E from pro-subtilisin E requires the removal of the N-terminal pro-sequence of 77 residues. Pro-subtilisin E produced in Escherichia coli using a pINIII-ompA vector was first extracted with 6 M guanidine-HCl and 5 M urea and purified to homogeneity in the presence of 5 M urea. Upon drop dialysis against 0.2 M sodium phosphate buffer (pH 6.2), the purified pro-subtilisin in 5 M urea was processed to active subtilisin of which the N-terminal sequence and migration in SDS-polyacrylamide gel electrophoresis were identical to those of authentic active subtilisin E. This process was found to be very sensitive to the ionic strengths and anions used. Under the optimum conditions (dialysis against 0.5 M (NH4)2SO4 and 1 mM CaCl2 in 10 mM Tris-HCl buffer (pH 7.0) at 4 degrees C for 1 h), approximately 20% of pro-subtilisin E was converted to active subtilisin E. The activation process was not inhibited by Streptomyces subtilisin inhibitor, and pro-subtilisin E in which the active site was mutated (Asp32 to Asn) was unable to be processed under the optimum conditions. These results confirmed the previous hypothesis that the processing of pro-subtilisin occurs by an intramolecular, autoprocessing mechanism.     

Cloning and analysis of WF146 protease, a novel thermophilic subtilisin-like protease with four inserted surface loops

Cloning and sequencing of the gene encoding WF146 protease, an extracellular subtilisin-like protease from the thermophile Bacillus sp. WF146, revealed that the WF146 protease was translated as a 416-amino acid precursor consisting of a putative 18-amino acid signal peptide, a 10-kDa N-terminal propeptide and a 32-kDa mature protease region. The mature WF146 protease shares a high degree of amino acid sequence identity with two psychrophilic subtilisins, S41 (68.2%) and S39 (65.4%), and a mesophilic subtilisin, SSII (67.1%). Significantly, these closely related proteases adapted to different temperatures all had four inserted surface loops not found in other subtilisins. However, unlike those of S41, S39 and SSII, the inserted loops of the WF146 protease possessed stabilizing features, such as the introduction of Pro residues into the loop regions. Interestingly, the WF146 protease contained five of the seven mutations previously found in a hyperstable variant of subtilisin S41 obtained by directed evolution. The proform of WF146 protease (pro-WF146 protease) was overexpressed in Escherichia coli in an inactive soluble form. After heat treatment, the 42-kDa pro-WF146 protease converted to a 32-kDa active mature form by processing the N-terminal propeptide. The purified mature WF146 protease hydrolyzed casein with an optimum temperature of 85 degrees C, and lost activity with a half-life of 30 min at 80 degrees C in the presence of 10 mM CaCl2.     

Folding pathway mediated by an intramolecular chaperone: propeptide release modulates activation precision of pro-subtilisin

Propeptides of several proteases directly catalyze the protein folding reaction. Uncatalyzed folding traps these proteases into inactive molten-globule-like conformers that switch into active enzymes only when their cognate propeptides are added in trans. Although tight binding and proteolytic susceptibility forces propeptides to function as single turnover catalysts, the significance of their inhibitory function and the mechanism of activation remain unclear. Using pro-subtilisin as a model, we establish that precursor activation is a highly coordinated process that involves synchronized folding, autoprocessing, propeptide release, and protease activation. Our results demonstrate that activation is controlled by release of the first free active protease molecule. This triggers an exponential cascade that selectively targets the inhibitory propeptide in the autoprocessed complex as its substrate. However, a mutant precursor that enhances propeptide release can drastically reduce the folding efficiency by altering the synergy between individual stages. Our results represent the first demonstration that propeptide release, not precursor folding, is the rate-determining step and provides the basis for the proposed model for precise spatial and temporal activation that allows proteases to function as regulators of biological function.     

Specific inhibition of mature fungal serine proteinases and metalloproteinases by their propeptides.

The function of the long propeptides of fungal proteinases is not known. Aspergillus fumigatus produces a 33-kDa serine proteinase of the subtilisin family and a 42-kDa metalloproteinase of the thermolysin family. These extracellular enzymes are synthesized as preproenzymes containing large amino-terminal propeptides. Recombinant propeptides were produced in Escherichia coli as soluble fusion proteins with glutathione S-transferase or thioredoxin and purified by affinity chromatography. A. fumigatus serine proteinase propeptide competitively inhibited serine proteinase, with a Ki of 5.3 x 10(-6) M, whereas a homologous serine proteinase from A. flavus was less strongly inhibited and subtilisin was not inhibited. Binding of metalloproteinase propeptide from A. fumigatus to the mature metalloenzyme was demonstrated. This propeptide strongly inhibited its mature enzyme, with a Ki of 3 x 10(-9) M, whereas thermolysin and a metalloproteinase from A. flavus were not inhibited by this propeptide. Enzymatically inactive metalloproteinase propeptide complex could be completely activated by trypsin treatment. These results demonstrate that the propeptides of the fungal proteinases bind specifically and inhibit the respective mature enzymes, probably reflecting a biological role of keeping these extracellular enzymes inactive until secretion.     

In vitro processing of pro-subtilisin produced in Escherichia coli

In a previous paper (Ikemura, H., Takagi, H., and Inouye, M. (1987) J. Biol. Chem. 262, 7859-7864), we demonstrated that the pro-sequence consisting of 77 amino acid residues at the amino terminus of subtilisin is essential for the production of active subtilisin. When the aggregates of pro-subtilisin produced in Escherichia coli were solubilized in 6 M guanidine hydrochloride and dialyzed against 200 mM sodium phosphate buffer (pH 7.1 or 6.2), pro-subtilisin was efficiently processed to active subtilisin. When more than 14 residues were removed from the amino terminus of the pro-sequence, active subtilisin was no longer produced as in the in vivo experiments. Similarly, active subtilisin would not renature under the same conditions once solubilized in guanidine hydrochloride. When the aspartic acid residue at the active site (Asp32) was altered to asparagine, processing of mutant pro-subtilisin was not observed even in the presence of wild-type pro-subtilisin. Inhibitors such as phenylmethanesulfonyl fluoride or Streptomyces subtilisin inhibitor did not block the processing of wild-type pro-subtilisin. These facts indicate that processing or pro-subtilisin is carried out by an intramolecular, self-processing mechanism. When the sample was dialyzed against 20 mM sodium phosphate (pH 6.2), no active subtilisin was found, suggesting that the highly charged nature of the pro-sequence plays an important role in the process of refolding of denatured pro-subtilisin.     

The autocatalytic processing of the subtilisin Carlsberg pro-region is independent of the primary structure of the cleavage site

Subtilisins are extracellular seryl-proteases produced by bacilli (Markland and Emil, 1971). In addition to signal sequences, these proteases have N-terminal extensions (pro-regions) which have also been identified in several other proteases (Silen et al., 1988; Vasantha et al., 1984; Polhner et al., 1987; Henderson et al., 1987; Yanagida et al., 1986; Takagi et al., 1985). The pro-region holds the pro-protease associated with the membrane and release of the protease takes place as a result of pro-region removal by autocatalytic processing (Egnell and Flock, 1991). In this report we describe the construction of four deletion-mutations in the gene encoding subtilisin Carlsberg at the junction between the pro-region and mature subtilisin Carlsberg. We found that the introduction of different deletions abolished the ability of subtilisin to undergo autocatalytic cleavage of the pro-region in cis, whereas cleavage by exogenous subtilisin could still occur in trans. Point mutations were also introduced in positions -5 to +4 around the pro-region and native subtilisin cleavage site. Processing of pro-subtilisin with the point mutations showed that the autocatalytic cleavage and recognition of this junction of the subtilisin Carlsberg pro-region is independent of the amino acid sequence around the cleavage site.     

In vitro stepwise autoprocessing of the proform of pro-aminopeptidase processing protease from Aeromonas caviae T-64

PA protease (pro-aminopeptidase processing protease) is an extracellular zinc metalloprotease produced by the Gram-negative bacterium Aeromonas caviae T-64. The 590-amino-acid precursor of PA protease is composed of a putative 19-amino-acid signal sequence, a 165-amino-acid N-terminal propeptide, a 33 kDa mature protease domain and an 11 kDa C-terminal propeptide. The proform of PA protease, which was produced as inclusion bodies in Escherichia coli, was subjected to in vitro refolding. It was revealed that the processing of the proform involved a stepwise autoprocessing mechanism. Firstly, the N-terminal propeptide was autocatalytically removed on completion of refolding and secondly, the C-terminal propeptide was autoprocessed after the degradation of the N-terminal propeptide. Both the N- and C-terminal propeptides existed as intact peptides after their successive removal, and they were subsequently degraded gradually. The degradation of the N-terminal propeptide appears to be the rate-limiting step in the maturation of the proform of PA protease.     

Characterization of an exo-beta-D-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

We previously clarified that the chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 produces diacetylchitobiose (GlcNAc(2)) as an end product from chitin. Here we sought to identify enzymes in T. kodakaraensis that were involved in the further degradation of GlcNAc(2). Through a search of the T. kodakaraensis genome, one candidate gene identified as a putative beta-glycosyl hydrolase was found in the near vicinity of the chitinase gene. The primary structure of the candidate protein was homologous to the beta-galactosidases in family 35 of glycosyl hydrolases at the N-terminal region, whereas the central region was homologous to beta-galactosidases in family 42. The purified protein from recombinant Escherichia coli clearly showed an exo-beta-D-glucosaminidase (GlcNase) activity but not beta-galactosidase activity. This GlcNase (GlmA(Tk)), a homodimer of 90-kDa subunits, exhibited highest activity toward reduced chitobiose at pH 6.0 and 80 degrees C and specifically cleaved the nonreducing terminal glycosidic bond of chitooligosaccharides. The GlcNase activity was also detected in T. kodakaraensis cells, and the expression of GlmA(Tk) was induced by GlcNAc(2) and chitin, strongly suggesting that GlmA(Tk) is involved in chitin catabolism in T. kodakaraensis. These results suggest that T. kodakaraensis, unlike other organisms, possesses a novel chitinolytic pathway where GlcNAc(2) from chitin is first deacetylated and successively hydrolyzed to glucosamine. This is the first report that reveals the primary structure of GlcNase not only from an archaeon but also from any organism.     

RNase I*, a form of RNase I, and mRNA degradation in Escherichia coli.

A previously unreported endoRNase present in the spheroplast fraction of Escherichia coli degraded homoribopolymers and small RNA oligonucleotides but not polymer RNA. Like the periplasmic endoRNase, RNase I, the enzyme cleaved the phosphodiester bond between any nucleotides; however, RNase I degraded polymer RNA as fast as homopolymers or oligomers. Both enzymes migrated as 27-kDa polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and could not be separated by various chromatographic procedures. In rna insertion mutants, both enzymes were completely missing; the spheroplast enzyme is called RNase I*, since it must be a form of RNase I. The two forms could be distinguished by physical treatments. RNase I could be activated by Zn2+, while RNase I* was inactive in the presence of Zn2+. RNase I was inactivated very slowly at 100 degrees C over a wide pH range, while RNase I* was inactivated slowly by heat at pH 4.0 but much more rapidly as the pH was increased to 8.0. In the presence of a thiol-binding agent, the inactivation at the higher pH values was much slower. These results suggest that RNase I*, but not RNase I, has free sulfhydryl groups. RNase I* activity in the cell against a common substrate was estimated to be several times that of RNase I. All four 2',3'-phosphomonoribonucleotides were identified in the soluble pools of growing cells. Such degradative products must arise from RNase I* activity. The activity would be suited for the terminal step in mRNA degradation, the elimination of the final oligonucleotide fragments, without jeopardizing the cell RNA. An enzyme with very similar specificity was found in Saccharomyces cerevisiae, suggesting that the activity may be widespread in nature.