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Peptidases or proteinases are now classified into seven families based on the nature of the catalytic residues [MEROPS-the peptidase database (http://merops.sanger.ac.uk/)]. They are aspartic- (first described in 1993), cysteine- (1993), serine- (1993) metallo- (1993), threonine- (1997), glutamic- (2004) and asparagine-peptidase (2010). By using an S-PI (pepstatin Ac) as a probe, a new subfamily of serine peptidase, serine-carboxyl peptidase (sedolisin) was discovered in 2001. In addition, the sixth family of peptidase, glutamic peptidase (eqolisin) was also discovered in 2004. The former peptidase is widely distributed in nature from archea to mammals, including humans. One of these enzymes is related to a human fatal hereditable disease, Batten disease. In contrast, the distribution of the latter peptidases is limited, with most of them found in human or plant pathogenic fungi. One such enzyme was isolated from a fungal infection in an HIV-infected patient. In this review, the background of the findings, and crystal structures, catalytic mechanisms, substrates specificities and distribution of the new peptidase families are described.

Kumamolisin-As is an acid collagenase with a subtilisin-like fold. Its active site contains a unique catalytic triad, Ser278-Glu78-Asp82, and a putative transition-state stabilizing residue, Asp164. In this study, the mutants D164N and E78H/D164N were engineered in order to replace parts of the catalytic machinery of kumamolisin-As with the residues found in the equivalent positions in subtilisin. Unlike the wild-type and D164N proenzymes, which undergo instantaneous processing to produce their 37-kDa mature forms, the expressed E78H/D164N proenzyme exists as an equilibrated mixture of the nicked and intact forms of the precursor. X-ray crystallographic structures of the mature forms of the two mutants showed that, in each of them, the catalytic Ser278 makes direct hydrogen bonds with the side chain of Asn164. In addition, His78 of the double mutant is distant from Ser278 and Asp82, and the catalytic triad no longer exists. Consistent with these structural alterations around the active site, these mutants showed only low catalytic activity (relative k(cat) at pH 4.0 1.3% for D164N and 0.0001% for E78H/D164N). pH-dependent kinetic studies showed that the single D164N substitution did not significantly alter the logk(cat) vs. pH and log(k(cat)/Km) vs. pH profiles of the enzyme. In contrast, the double mutation resulted in a dramatic switch of the logk(cat) vs. pH profile to one that was consistent with catalysis by means of the Ser278-His78 dyad and Asn164, which may also account for the observed ligation/cleavage equilibrium of the precursor of E78H/D164N. These results corroborate the mechanistic importance of the glutamate-mediated catalytic triad and oxyanion-stabilizing aspartic acid residue for low-pH peptidase activity of the enzyme.

Kumamolisin, a serine carboxyl proteinase, is very stable and hardly denatured by single perturbation of a chemical denaturant (urea), pressure (<500 MPa) or temperature (<65 degrees C). In order to investigate the cooperative effects of these three denaturing agents, DSC, CD, intrinsic fluorescence, and fourth derivative UV absorbance were measured under various conditions. By application of pressure to kumamolisin in 8 M urea solution, substantial red-shift in the center of fluorescence emission spectral mass was observed, and the corresponding blue-shift was observed for two major peaks in fourth derivative UV absorbance, under the similar urea-containing conditions. The denaturation curves were analyzed on the basis of a simple two-state model in order to obtain thermodynamic parameters (DeltaV, DeltaG, and m values), and the combined effects of denaturing agents are discussed, with the special interest in the large cavity and neighboring Trp residue in kumamolisin.

Kumamolisin, an extracellular proteinase derived from an acido/thermophilic Bacillus, belongs to the sedolisin family of endopeptidases characterized by a subtilisin-like fold and a Ser-Glu-Asp catalytic triad. In kumamolisin, the Asp82 carboxylate hydrogen bonds to Glu32-Trp129, which might act as a proton sink stabilizing the catalytic residues. The 1.2/1.3 A crystal structures of the Glu32-->Ala and Trp129-->Ala mutants show that both mutations affect the active-site conformation, causing a 95% activity decrease. In addition, the 1.2 A crystal structure of the Ser278-->Ala mutant of pro-kumamolisin was determined. The prodomain exhibits a half-beta sandwich core docking to the catalytic domain similarly as the equivalent subtilisin prodomains in their catalytic-domain complexes. This pro-kumamolisin structure displays, for the first time, the uncleaved linker segment running across the active site and connecting the prodomain with the properly folded catalytic domain. The structure strongly points to an initial intramolecular activation cleavage in subtilases, as presumed for pro-subtilisin and pro-furin.

Kumamolisin-As (previously called ScpA) is the first known example of a collagenase from the sedolisin family (MEROPS S53). This enzyme is active at low pH and in elevated temperatures. In this study that used x-ray crystallographic and biochemical methods, we investigated the structural basis of the preference of this enzyme for collagen and the importance of a glutamate residue in the unique catalytic triad (Ser(278)-Glu(78)-Asp(82)) for enzymatic activity. Crystal structures of the uninhibited enzyme and its complex with a covalently bound inhibitor, N-acetyl-isoleucyl-prolyl-phenylalaninal, showed the occurrence of a narrow S2 pocket and a groove that encompasses the active site and is rich in negative charges. Limited endoproteolysis studies of bovine type-I collagen as well as kinetic studies using peptide libraries randomized at P1 and P1', showed very strong preference for arginine at the P1 position, which correlated very well with the presence of a negatively charged residue in the S1 pocket of the enzyme. All of these features, together with those predicted through comparisons with fiddler crab collagenase, a serine peptidase, rationalize the enzyme's preference for collagen. A comparison of the Arrhenius plots of the activities of kumamolisin-As with either collagen or peptides as substrates suggests that collagen should be relaxed before proteolysis can occur. The E78H mutant, in which the catalytic triad was engineered to resemble that of subtilisin, showed only 0.01% activity of the wild-type enzyme, and its structure revealed that Ser(278), His(78), and Asp(82) do not interact with each other; thus, the canonical catalytic triad is disrupted.

The molecular structure of the pepstatin-insensitive carboxyl peptidase from Scytalidium lignicolum, formerly known as scytalidopepsin B, was solved by multiple isomorphous replacement phasing methods and refined to an R factor of 0.230 (R(free) = 0.246) at 2.1-A resolution. In addition to the structure of the unbound peptidase, the structure of a product complex of cleaved angiotensin II bound in the active site of the enzyme was also determined. We propose the name scytalidocarboxyl peptidase B (SCP-B) for this enzyme. On the basis of conserved, catalytic residues identified at the active site, we suggest the name Eqolisin for the enzyme family. The previously uninvestigated SCP-B fold is that of a beta-sandwich; each sheet has seven antiparallel strands. A tripeptide product, Ala-Ile-His, bound in the active site of SCP-B has allowed for identification of the catalytic residues and the residues in subsites S1, S2, and S3, which are important for substrate binding. The most likely hydrolytic mechanism involves nucleophilic attack of a general base (Glu-136)-activated water (OH(-)) on the si-face of the scissile peptide carbonylcarbon atom to form a tetrahedral intermediate. Electrophilic assistance and oxyanion stabilization is provided by the side-chain amide of Gln-53. Protonation of the leaving-group nitrogen is accomplished by the general acid function of the protonated carboxyl group of Glu-136.

Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest. Alicyclobacillus sendaiensis strain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides. Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA. Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined. On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala*Gly-Pro-Ile-Gly (k(cat), 5.41 s(-1); K(m), 32 micro M) and Met-Gly-Pro-Arg*Gly-Phe-Pro-Gly-Ser (k(cat), 351 s(-1); K(m), 214 micro M), where the asterisks denote the scissile bonds. The cloned scpA gene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da. Heterologous expression of the scpA gene in the Escherichia coli cells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein. Sequencing of the scpA gene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results. Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined.

Kumamolysin is a thermostable endopeptidase from Bacillus novosp. MN-32, exhibiting maximal proteolytic activity around pH 3. It belongs to the newly identified family of serine-carboxyl proteinases, which also includes CLN2, a human lysosomal homolog recently implicated in a fatal neurodegenerative disease. Kumamolysin and its complexes with two aldehyde inhibitors were crystallized, and their three-dimensional structures were solved and refined with X-ray data to 1.4 A resolution. As its Pseudomonas homolog, kumamolysin exhibits a Ser/Glu/Asp catalytic triad with particularly short interconnecting hydrogen bonds and an oxyanion hole enabling the reactive serine to attack substrate peptide bonds at quite acidic pH. An additional Glu/Trp pair, unique to kumamolysin, might further facilitate proton delocalization during nucleophilic attack, in particular at high temperature.

The gene encoding kumamolysin, a thermostable pepstatin-insensitive carboxyl proteinase, was cloned and expressed. (i) Kumamolysin was synthesized as a large precursor consisting of two regions: amino-terminal prepro (188 amino acids) and mature proteins (384 amino acids). (ii) The deduced amino acid sequence of the mature region exhibited high similarity to those of such bacterial pepstatin-insensitive enzymes as Pseudomonas carboxyl proteinase (PSCP; EC 3.4.23.37, identity = 37%), Xanthomonas carboxyl proteinase (XCP; EC 3.4.23.33, identity = 36%), and human CLN2 gene product (identity = 36%), which is related to a fatal neurodegenerative disease. (iii) The presumed catalytic triad, Glu78, Asp82, Ser278 [three-dimensional structure of PSCP: Wlodawer, A. et al. (2001) Nature Struct. Biol., 8, 442-446], was found to be conserved in the amino acid sequence of kumamolysin. (iv) Kumamolysin was inactivated by such aldehyde-type inhibitors as Ac-Ile-Pro-Phe-CHO (K(i) = 0.7 0.14 microM). In PSCP, it has been clarified that these inhibitors form a hemiacetal linkage with the catalytic serine residue and inactivate the enzyme. (v) Mutational analysis of the Ser278 residue revealed that the mutant lost both auto-processing activity and proteolytic activity. These results strongly suggest that kumamolysin has a unique catalytic triad consisting of Glu78, Asp82, and Ser278 residues, as previously observed for PSCP.

The pressure dependence of the activity and spectroscopic properties of four carboxyl proteinases were investigated. Two were pepstatin-sensitive carboxyl proteinases (porcine pepsin and proteinase A from baker's yeast) and two were pepstatin-insensitive carboxyl proteinases (from Pseudomonas sp. 101 (pseudomonapepsin; PCP) and Xanthomonas sp. T-22 (xanthomonapepsin; XCP)). The specificity constant [k(cat)/K(m(app))] of PCP and XCP for a synthetic peptide substrate showed only a slight decrease with increasing pressure, whereas pepsin and proteinase A showed substantial disactivation at higher pressures. The calculated apparent activation volume (Delta V((k(cat)/(K(m)) was about 1, 3, 13, and 14 mL.mol(-1) for PCP, XCP, pepsin, and proteinase A, respectively. The hydrolysis of acid-denatured myoglobin by the four carboxyl proteinases was only slightly affected by high pressure (except for proteinase A at 400 MPa), in contrast to the results for the peptide hydrolysis. In fact, PCP, XCP, and proteinase A actually showed slightly higher degradations of acid-denatured myoglobin at higher pressures. The residual activities of these enzymes after the incubation at high pressures implied a pressure-induced stabilization towards autolysis. The changes in the fourth derivative near-UV absorbance spectrum of the four enzymes in aqueous solution were measured at various pressures from 0.1 to 400 MPa. Upon an increase in pressure, the peaks from PCP and XCP red-shifted slightly, whereas pepsin and proteinase A blue-shifted substantially, thus indicating a more polar environment. The intrinsic fluorescence also decreased upon increasing pressure. However, the change for XCP was rather small, but the change for the other three was very large. The changes in the peak wavelength for pepsin and proteinase A were characteristic, and also indicated a more polar environment under high pressure. An analysis by the center of spectra mass (CSM) gave the Delta G and Delta V of transition as 9.8 kJ x mol(-1) and -24 mL x mol(-1) (pepsin) and 11.7 kJ x mol(-1) and -43 mL x mol(-1) (proteinase A), respectively, by assuming a simple two-state transition. The circular dichroism (CD) showed relatively small changes after 1-h incubations at 400 MPa, indicating that the secondary structures were largely maintained.

Kumamolysin, a carboxyl proteinase from Bacillus novosp. MN-32, is characterized by its thermostability and insensitivity to aspartic proteinase inhibitors such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3-(p-nitro-phenoxy)propane. Here, its substrate specificity was elucidated using two series of synthetic chromogenic substrates: P(5)-P(4)-P(3)-P(2)-Phe*Nph (p-nitrophenylalanine: *cleavage site)-P(2)'-P(3)', in which the amino acid residues at the P(5)-P(2), P(2)' and P(3)' positions were systematically substituted. Among 74 substrates, kumamolysin was shown to hydrolyze Lys-Pro-Ile-Pro-Phe-Nph-Arg-Leu most effectively. The kinetic parameters of this peptide were K(m) = 41+/-5 microM, k(cat) = 176+/- 10 s(-1), and k(cat)/K(m) = 4.3+/-0.6 mM(-1) x s(-1). These systematic analyses revealed the following features: (i) Kumamolysin had a unique preference for the P(2) position. Kumamolysin preferentially hydrolyzed peptides having an Ala or Pro residue at the P(2) position; this was also observed for the pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4 [J-4; Shibata et al. (1998) J. Biochem. 124, 642-647]. Other carboxyl proteinases, including Pseudomonas sp. 101 pepstatin-insensitive carboxyl proteinase (PCP) and Xanthomonas sp. T-22 pepstatin-insensitive carboxyl proteinase (XCP), preferred peptides having hydrophobic and bulky amino acid residue such as Leu at the P(2) position. (ii) Kumamolysin preferred such charged amino acid residues as Glu or Arg at the P(2)' position, suggesting that the S(2)' subsite of kumamolysin is occupied by hydrophilic residues, similar to that of PCP, XCP, and J-4. In general, the S(2)' subsite of pepstatin-sensitive carboxyl proteinases (aspartic proteinases) is hydrophobic in nature. Thus, the hydrophilic nature of the S(2)' subsite was confirmed to be a distinguishing feature of pepstatin-insensitive carboxyl proteinases from prokaryotes.

An expression and secretion system for scytalidopepsin B, an acid protease from Scytalidium lignicolum, was constructed in yeast. Saccharomyces cerevisiae AH22 was transformed with an yeast-E. coli shuttle vector, pAM82, in which an yeast invertase signal segment and the cDNA encoding the pro- and mature enzyme regions were inserted. The transformant was found to secret a pepstatin-insensitive acid protease, when cultured aerobically in a low phosphate (Pi) medium. Amino terminal amino acid sequencing analysis indicated that the recombinant acid protease was accurately processed and secreted as a mature form.

Pepstatin-insensitive carboxyl proteinases from Pseudomonas sp. (PCP) and Xanthomonas sp. (XCP) have no conserved catalytic residue sequences, -Asp*-Thr-Gly- (Asp is the catalytic residue) for aspartic proteinases. To identify the catalytic residues of PCP and XCP, we selected presumed catalytic residues based on their high sequence similarity, assuming that such significant sites as catalytic residues will be generally conserved. Several Ala mutants of Asp or Glu residues were constructed and analyzed. The D170A, E222A, and D328A mutants for PCP and XD79A, XD169A, and XD348A mutants for XCP were not converted to mature protein after activation, and no catalytic activity could be detected in these mutants. The specificity constants toward chromogenic substrate of the other PCP and XCP mutants, except for the D84A mutant of PCP, were similar to that of wild-type PCP or XCP. Coupled with the result of chemical modification (Ito, M., Narutaki, S., Uchida, K., and Oda, K. (1999) J. Biochem. (Tokyo) 125, 210-216), a pair of Asp residues (170 and 328) for PCP and a pair of Asp residues (169 and 348) for XCP were elucidated to be their catalytic residues, respectively. The Glu(222) residue in PCP or Asp(79) residue in XCP was excluded from the candidates as catalytic residues, since the corresponding mutant retained its original activity.

Pseudomonas carboxyl proteinase (PCP), isolated from Pseudomonas sp. 101, is the first example from a prokaryote of unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to aspartic proteinase inhibitors, such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3(p-nitrophenoxy)propane. To identify the catalytic residue(s) of PCP, chemical modification was carried out using carboxyl residue-specific reagents, carbodiimides. PCP was inactivated effectively by N,N'-dicyclohexylcarbodiimide (DCCD) with pseudo-first-order kinetics. For the inactivation, 0.7 mol DCCD was involved per 1 mol PCP. The effects of pH and methanol on the inactivation showed that two carboxyl residues (Asp and/or Glu) were involved in the reaction. The inactivation by DCCD was prevented by a competitive inhibitor, tyrostatin, or a synthetic substrate in a concentration-dependent manner. Based on these data, differential labeling of PCP with DCCD was carried out: Firstly, PCP was treated with cold DCCD in the presence of tyrostatin. After removal of the tyrostatin, which covered the substrate binding site, by dialysis, the PCP was treated with [14C]DCCD to label carboxyl residue(s) essential for its function. Two labeled peptides were isolated by HPLC from a trypsin digest of cold- and [14C]DCCD modified PCP. On analysis of their amino acid sequences, it was revealed that the [14C]DCCD was bound to Asp140 and Glu222 of PCP, respectively. Based on these data, it was strongly suggested that Asp140 and Glu222 of PCP were involved in its catalytic function or substrate binding.

Pseudomonas sp. 101 carboxyl proteinase (PCP) and Xanthomonas sp. T-22 carboxyl proteinase (XCP), the first and second unique carboxyl proteinases from prokaryotes to be isolated and characterized, are not inhibited by the classical carboxyl proteinase inhibitor pepstatin. In this study, we elucidated their subsite preferences by using a series of synthetic chromogenic substrates, Lys-Pro-Ile(P3)-Glu(P2)-Phe*Nph-Arg(P2')-Leu(P3') (Nph is p-nitrophenylalanine, Phe*Nph is the cleavage site) with systematic substitutions at the P3, P2, P2', and P3' positions. Among 45 substrates tested, the best substrate for PCP had a Leu replacement at the P2 position (kcat = 27.2 s-1, Km = 4.22 microM, kcat/Km = 6.43 microM-1.s-1), and that for XCP had an Ala replacement at the P3 position (kcat = 79.4 s-1, Km = 6.05 microM, kcat/Km = 13.1 microM-1. s-1). PCP and XCP preferred such charged amino acid residues as Glu, Asp, Arg, or Lys at the P2' position. This suggested that the S2' subsites of PCP and XCP are occupied by hydrophilic residues, similar to that of pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4 [Shibata et al. (1998) J. Biochem. 124, 642-647]. In contrast, the S2' subsite of pepstatin-sensitive carboxyl proteinases (aspartic proteinases) is hydrophobic in nature. Thus, the hydophilic nature of the S2' subsite appears to be a distinguishing feature of pepstatin-insensitive carboxyl proteinases.

A chromosomal DNA of Scytalidium lignicolum was digested with Sau3AI. The digest was self-ligated and amplified by inverse PCR procedure using primers designed based on the nucleotide sequences of up- and down-stream regions of an intron present in the scytalidopepsin B gene. Analysis of the nucleotide sequence of PCR product (700 bp) showed that the enzyme is synthesized as a precursor protein consisting of the prepro- and mature enzyme regions. The deduced amino acid sequence was highly similar to those of aspergillopepsin A and recently reported endothiapepsins B and C, but quite different from those of pepstatin-insensitive bacterial acid proteases and the pepstatin-sensitive aspartic protease family.

The substrate specificities and kinetic properties of proteinase A, an intracellular aspartic proteinase from the yeast Saccharomyces cerevisiae, were determined using a series of synthetic chromogenic peptides with the general structure P5-P4-P3-P2-Phe-(NO2)Phe-P2'-P3' [P5, P4, P3, P2, P2', P3' are various amino acids; (NO2)Phe is p-nitro-L-phenylalanine]. The nature of the residues occupying the NH2-terminal region of the substrate had a strong influence on the kinetic constants. Among those tested, Ala-Pro-Ala-Lys-Phe-(NO2)-Phe-Arg-Leu had the best kinetic constants (Km = 0.012 mM, kcat = 14.4 s-1, kcat/Km = 1,200 M-1.s-1). Compared with such aspartic proteinases as pepsin, cathepsin D, and renin, the substrate specificity of proteinase A was unique. Based on these results, a novel fluorescent substrate, MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH2, was developed for the sensitive measurement of proteinase A.

Pseudomonas carboxyl proteinase (PCP), isolated from Pseudomonas sp. 101, and Xanthomonas carboxyl proteinase (XCP), isolated from Xanthomonas sp. T-22, are the first and second examples of unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to aspartic proteinase inhibitors, such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3(p-nitrophenoxy)propane. The substrate specificities of PCP and XCP were studied using a series of synthetic chromogenic peptide substrates with the general structure, P5-P4-P3-P2-Phe-Nph-P2'-P3' (P5, P4, P3, P2, P2', P3': a variety of amino acids, Nph is p-nitro-L-phenylalanine, and the Phe-Nph bond is cleaved). PCP and XCP were shown to hydrolyze a synthetic substrate, Lys-Pro-Ala-Leu-Phe-Nph-Arg-Leu, most effectively among 28 substrates. The kinetic parameters of this peptide for PCP were Km = 6.3 microM, Kcat = 51.4 s-1, and kcat/Km = 8.16 microM-1.s-1. The kinetic parameters for XCP were Km = 3.6 microM, kcat = 52.2 s-1, and kcat/Km = 14.5 microM-1.s-1. PCP showed a stricter substrate specificity than XCP. That is, the specificity constant (kcat/Km) of each substrate for PCP was in general < 0.5 microM-1.s-1, but was drastically improved by the replacement of Lys by Leu at the P2 position. On the other hand, XCP showed a less stringent substrate specificity, with most of the peptides exhibiting reasonable kcat/Km values (> 1.0 microM-1.s-1). Thus it was found that the substrate specificities of PCP and XCP differ considerably, in spite of the high similarity in their primary structures. In addition, tyrostatin was found to be a competitive inhibitor for XCP, with a Ki value of 2.1 nM, as well as for PCP (Ki = 2.6 nM).

Xanthomonas carboxyl proteinase (XCP), isolated from Xanthomonas sp. T-22, is the second example of the unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to the classical aspartic proteinase inhibitor. The gene coding for XCP was cloned, sequenced, and expressed in Escherichia coli. The XCP gene contains an open reading frame of 2,481 base pairs encoding a protein of 827 amino acid residues with a M(r) of 83,677. The XCP was synthesized as a large precursor consisting of three regions: NH2-terminal prepro (N-Prepro) (237 amino acid residues); mature XCP (398 a.a.residues); and COOH-terminal pro (C-Pro) (192 a.a. residues). The N-Prepro and mature XCP regions had no sequence similarity to any other proteins reported so far, except the carboxyl proteinase from Pseudomonas sp. 101 [Oda, K., Takahashi, T., Tokuda, Y., Shibano, Y., and Takahashi, S. (1994) J. Biol. Chem. 269, 26518-26524]. The C-Pro region showed high similarity to COOH-terminal regions of other microbial proteinase precursors. E. coli carrying a plasmid containing the cloned wild-type XCP gene produced an 84-kDa protein. This protein was processed into a mature, active form under acidic conditions. This process was completely blocked by tyrostatin, an XCP-specific inhibitor from Kitasatosporia sp. 55, indicating an autocatalytic processing. The purified recombinant XCP had the same characteristics as authentic XCP except for the NH2-terminal amino acid sequence. When the mutant XCP gene truncated in the C-Pro region was expressed in E. coli, an expected 64-kDa protein was detected in the cells, and also processed into the 42-kDa active form under the acidic conditions. Thus, the C-Pro region was not essential for the formation of active mature XCP.

A chromosomal DNA fragment of Scytalidium lignicolum that encodes the mature enzyme region of acid protease B (Scytalidopepsin B), was cloned and its nucleotides sequenced. The fragment contained a 76-bp intron at the middle of the mature enzyme-coding region. The mature enzyme was composed of 206 amino acid residues with a molecular weight of 21,550. There were some discrepancies between the amino acid sequence deduced from these results and that previously established by protein sequencing.

A unique carboxyl proteinase (EC 3.4.23.33), insensitive to the classical inhibitor isovaleryl pepstatin and isolated from Pseudomonas sp. 101 (PCP), is the first example of a prokaryotic enzyme of this class. The gene coding for PCP was cloned, sequenced, and expressed in Escherichia coli. The gene consists of 1,761 base pairs encoding a protein of 587 amino acid residues. The NH2-terminal 215-amino acid preprosequence flanks the 372-amino acid mature protein, which is identical with the primary structure of an authentic PCP determined by chemical methods. E. coli carrying a plasmid containing the cloned wild-type PCP gene produced a 62-kDa protein. This molecule was processed and secreted into the periplasm as a 43-kDa protein, which converted to mature PCP under acidic conditions. This autocatalytic conversion was completely blocked by tyrostatin, a PCP-specific peptidic inhibitor from Kitasatosporia sp. 55. The purified recombinant PCP has the same characteristics as authentic PCP. When several preprosequence deletion mutants were expressed in E. coli, mutant proteins were accumulated as insoluble forms with no proteinase activities. These results suggest that the prepropeptide of PCP plays an essential role in the formation of functional PCP.

We have found a novel type of thermostable, pepstatin-insensitive carboxyl proteinase in the culture filtrate of Bacillus novosp. MN-32. The carboxyl proteinase, which was named kumamolysin, was purified about 8,300-fold by column chromatography including DEAE-Sepharose CL-6B, Sephadex G-100, and TSKgel DEAE-5PW. The purified kumamolysin gave a single band corresponding to 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of kumamolysin was estimated to be 40 kDa by gel filtration. The isoelectric point of kumamolysin was estimated to be pH 3.5 by isoelectric focusing. Kumamolysin has maximum proteolytic activity at 70 degrees C and at pH 3.0. Kumamolysin specifically hydrolyzed the Leu15-Tyr16 peptide bond in oxidized insulin B-chain (Km = 9.0 x 10(-5) M, Kcat = 71 s-1; at pH 3.0, 30 degrees C), and additional cleavage at Phe25-Tyr26 was detected at a considerably lower rate. Kumamolysin is insensitive to the known carboxyl proteinase inhibitors pepstatin, diazoacetyl-DL-norleucine methyl ester, and 1,2-epoxy-3-(p-nitrophenoxy)propane. Kumamolysin has no similarity to the thermostable acid protease thermopsin from Sulfolobus acidocaldarius (Lin, X.-L., and Tang, J. (1990) J. Biol. Chem. 265, 1490-1495). Thus, the substrate specificity, the inhibitor sensitivity, the molecular mass, and the thermostability all suggest that kumamolysin is a novel type of carboxyl proteinase.

The acid protease B (SLB) of Scytalidium lignicolum was reduced and carboxymethylated and then subjected to tryptic digestion. Five fragments were isolated and some of them were further digested with alpha-chymotrypsin, thermolysin, and dilute acetic acid. The sequence analysis of these fragments and the peptides by conventional methods established the complete amino acid sequence of SLB. The enzyme was composed of 204 amino acid residues with threonine and valine as its amino- and carboxyl-termini, respectively. Locations of three disulfide bridges were also established to be Cys47-126, Cys140-163, and Cys192-201 by enzymatic fragmentation of the denatured and unmodified SLB. Only a slight homology was found in the sequences of SLB and other acid proteases hitherto reported.

Scytalidium lignicolum acid protease B, a pepstatin-insensitive acid protease, was modified by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) with the concomitant loss of its enzyme activity, and an EPNP-labeled peptide was isolated from the thermolysin-digest of the modified enzyme by HPLC. The amino acid sequence of the peptide was determined to be Ile-Leu-Glu-Thr-Gly, which corresponds to the sequence of residue Nos. 51-55 of the enzyme. The results of treatment of the labeled peptide with hydroxylamine suggested that the EPNP moiety is ester-linked to Glu53 of the enzyme. The amino acid sequence around Glu53 of the acid protease B showed high homology with those around the active site Asp residues of calf chymosin and porcine pepsin. These results show that it is highly possible that Glu53 of the acid protease B is one of the amino acid residues involved in its catalytic activity.