Cloning and rational mutagenesis of kexstatin I, a potent proteinaceous inhibitor of Kex2 proteinase.

Kexstatin I is a potent proteinaceous inhibitor of Kex2 proteinase (EC 3.4.21.61). In the present study we show the molecular cloning, primary structure determination and expression of the gene encoding kexstatin I. We also demonstrate its enhanced activity and specificity for Kex2 proteinase inhibition by rational mutagenesis. The cloned kexstatin I gene encoded a protein of 145 amino acid residues, including the 35-residue signal sequence for secretion. The amino acid sequence showed 52% identity with those of the Streptomyces subtilisin inhibitors (SSIs). Thus kexstatin I is the first SSI-family member that can inhibit Kex2 proteinase. The reactive site of the inhibitor was determined to be -Thr(69)-Lys(70) downward arrowGlu(71)-, where downward arrow indicates the reactive site. Because Kex2 proteinase generally shows the highest affinity for substrates with basic amino acid residues at the P(1) and P(2) sites, conversion of the Thr(69)-Lys(70) segment of the inhibitor into dibasic motifs was expected to result in enhanced inhibitory activities. Thus we constructed kexstatin I mutants, in which the Thr(69)-Lys(70) sequence was replaced by the Thr(69)-Arg(70), Lys(69)-Lys(70) and Lys(69)-Arg(70) sequences using PCR-based mutagenesis, and analysed them kinetically. Among these mutants, the Lys(69)-Arg(70) mutant was the most potent inhibitor. The K(i) for Kex2 proteinase was 3.2x10(-10) M, which was 140-fold lower than that of the inhibitor with the Thr(69)-Lys(70) sequence. Although kexstatin I could also inhibit subtilisin, the enhancement of inhibitory activity upon such mutations was specific for Kex2 proteinase inhibition.

An active-site mutation causes enhanced reactivity and altered regiospecificity of transglucosylation catalyzed by the Bacillus sp. SAM1606 alpha-glucosidase.

Bacillus sp. SAM1606 alpha-glucosidase catalyzes the transglucosylation of sucrose to produce three regioisomers of the glucosylsucroses, with theanderose (6-O(G)-glucosylsucrose) as the most abundant transfer product. To find the active-site amino acid residues which can affect the reactivity and regiospecificity of the glucosyl transfer, 16 mutants with amino acid substitutions near the active site were allowed to react with 1.75 M sucrose at 60 degrees C, pH 6.0, and the course of transglucosylation as well as the product specificity were analyzed. The sites of the amino acid substitutions were selected by comparing the conserved amino acid sequences located near the active site of the SAM1606 enzyme with those of the Bacillus oligo-1,6-glucosidases (O16G), which have very high amino acid sequence similarities near the active site but have a distinct substrate specificity. The results showed that, among the mutated SAM1606 enzymes examined, only the mutants with substitution of Gly273 with Pro showed an altered reactivity and specificity of transglucosylation; these mutants exhibited a significantly enhanced initial velocity of glucosyl transfer, yielding isomelezitose (6-O(F)-glucosylsucrose) instead of theanderose as the major transfer product. These results indicate that the substitution of Gly273 with Pro critically governs the enhanced reactivity and altered specificity of the transglucosylation. The notion that the amino acid residue at this position is the determinant of the glucosyl-transfer specificity was further confirmed by observation that the Bacillus cereus O16G, which has a proline at the corresponding position, produced isomelezitose as the major transfer product during transglucosylation with sucrose.

Mutational analysis of the role of His452 of Saccharopolyspora rectivirgula beta-galactosidase.

To examine the role of His452 of the Saccharopolyspora rectivirgula beta-galactosidase in the binding of a tightly bound, catalytically important Mn2+ (i.e., class II Mn2+) ion, His452 was replaced with Phe or Glu and the respective site-directed mutants, H452F and H452E, were characterized. Neither mutant contained Mn2+ in an Mn2+-free buffer and both were virtually inactive in the absence of Mn2+ (their relative activities being less than 0.03% that of the fully activated wild-type enzyme). When Mn2+ was added, however, the mutants were activated to 3% (for H452F) and 0.8% (for H452E) of the full activity of the wild type. The Mn2+ concentrations needed for half-maximal activation of H452F and H452E were, respectively, 15,000 and 5000 times higher than the reported dissociation constant (2 nM) of the class II Mn2+, suggesting that His452 plays a key role in the binding of this catalytically important Mn2+. Activation of the mutants by Mn2+, albeit very weak, contrasts with a lack of any such metal activation previously observed with the two corresponding mutants of Escherichia coli lacZ beta-galactosidase.

Unique primary structure of a thermostable multimetal beta-galactosidase from Saccharopolyspora rectivirgula.

The gene of the monomeric multimetal beta-galactosidase of Saccharopolyspora rectivirgula was cloned and sequenced. Although the enzyme could be assigned as a member of beta-galactosidases belonging to the glycosyl hydrolase family 2, it has unusual structural features for beta-galactosidase of this family; it contained a unique sequence which consists of approximately 200 amino acid residues with no similarity to known proteins. This 200-residue sequence exists as if it is inserted into a sequence homologous to the active-site domain of the Escherichia coli lacZ enzyme.

Substrate specificities and kinetic properties of proteinase A from the yeast Saccharomyces cerevisiae and the development of a novel substrate.

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.

Long-range effect of mutation of calcium binding aspartates [correction of asparates] on the catalytic activity of alkaline protease from Pseudomonas aeruginosa.

Apart from a catalytic domain, the alkaline protease of Pseudomonas aeruginosa has a novel parallel beta-helix domain stabilized through Ca2+ binding. In order to clarify the importance of the beta-helix structure in maturation of the enzyme, aspartic residue D-356 or D-365 in the Ca2+ binding sequence motif was replaced with L-alanine, and the catalytic activity of each mutant was assayed. These mutants did not show any proteolytic activity, although the composition of their polypeptide chains was the same as that of the wild type except for the mutated alanine residue. These results suggest that D-356 and D-365 are important in control of the beta-helix folding induced by Ca2+ binding and that incomplete beta-helix folding due to the lack of their side-chains affects the maturation of the enzyme in the long-range order.

Altering substrate specificity of Bacillus sp. SAM1606 alpha-glucosidase by comparative site-specific mutagenesis.

The Bacillus sp. SAM1606 alpha-glucosidase with a broad substrate specificity is the only known alpha-glucosidase that can hydrolyze alpha,alpha'-trehalose efficiently. The enzyme exhibits a very high sequence similarity to the oligo-1,6-glucosidases (O16G) of Bacillus thermoglucosidasius and Bacillus cereus which cannot act on trehalose. These three enzymes share 80% identical residues within the conserved regions (CR), which have been suggested to be located near or at the active site of the alpha-amylase family enzymes. To identify by site-specific mutagenesis the critical residues that determine the broad substrate specificity of the SAM1606 enzyme we compared the CR sequences of these three glucosidases and selected five targets to be mutagenized in SAM1606 alpha-glucosidase, Met76, Arg81, Ala116, Gly273, and Thr342. These residues have been specifically replaced by in vitro mutagenesis with Asn, Ser, Val, Pro, and Asn, respectively, as in the Bacillus O16G. The 12 mutant enzymes with single and multiple substitutions were expressed and characterized kinetically. The results showed that the 5-fold mutation virtually abolished the affinity of the enzyme for alpha, alpha'-trehalose, whereas the specificity constant for the hydrolysis of isomaltose, a good substrate for both the SAM1606 enzyme and O16G, remained essentially unchanged upon the mutation. This loss in affinity for trehalose was critically governed by a Gly273 --> Pro substitution, whose effect was specifically enhanced by the Thr342 --> Asn substitution in the 5-fold and quadruple mutants. These results provide evidence for the differential roles of the amino acid residues in the CR in determining the substrate specificity of the alpha-glucosidase.

Cloning and expression of an isovaleryl pepstatin-insensitive carboxyl proteinase gene from Xanthomonas sp. T-22.

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 novel proteinaceous Kex 2 proteinase inhibitor, kexstatin, from Streptomyces platensis Q268.

We found a novel proteinaceous Kex 2 proteinase inhibitor, named kexstatin, in the culture supernatant of Streptomyces platensis Q268. The purified kexstatin was homogeneous by SDS-PAGE and the molecular weight was estimated to be 13,000. The N-terminal amino acid sequence of kexstatin has high similarity to Streptomyces subtilisin inhibitor (SSI), suggesting that kexstatin belongs to the SSI family. Kexstatin was a strong inhibitor of Kex 2 proteinase and subtilisin but not thermolysin, trypsin, or chymotrypsin. The IC50 value of kexstatin against 1 microgram of Kex 2 proteinase was 1.4 micrograms.

High-level secretion of fungal glucoamylase using the Candida boidinii gene expression system.

The methylotrophic yeast, Canadida boidinii, was investigated as an expression host for secretory enzyme production. The cDNA of Rhizopus oryzae glucoamylase was placed under the C. boidinii alcohol oxidase (AODl) promoter. A transformant integrated with a single-copy expression cassette to the chromosome produced glucoamylase into the medium to a high amount when the cells were grown on methanol or methanol plus glycerol as (a) carbon source(s). The transformant C. boidinii cells were grown up to ca. 95 g dry cell weight/liter medium, and the concentration of glucoamylase in the medium reached 3.4 g/liter. This showed that the signal sequence from Rhizopus glucoamylase functioned very efficiently in C. boidinii. Next, secreted glucoamylase from C. boidinii was purified and compared with the enzyme produced in S. cerevisiae. The enzyme produced in C. boidinii was found to have higher molecular weight than that produced in S. cerevisiae, which was due to the difference of the N-linked glycosylated sugar structure of the produced proteins.

Single point mutations in Met4p impair the transcriptional repression of MET genes in Saccharomyces cerevisiae.

Transcription of MET genes in Saccharomyces cerevisiae depends on a transcriptional activator, the MET4 gene product (Met4p). Using in vitro mutagenesis, we isolated two mutant MET4 alleles encoding [Pro215]Met4p and [Ser156]Met4p. These mutations impeded Met4p's responsiveness to methionine in the media, and yeast cells carrying mutant alleles exhibited enhanced transcription of MET genes under repressing conditions. The enhanced transcription was dependent on the CBF1 gene, but did not compete with an excess of wild-type Met4p, suggesting that some changes in the affinity of Met4p to other factors might be involved in S-adenosylmethionine-mediated transcriptional regulation.

Cloning, sequencing, and heterologous expression of a gene coding for Arthromyces ramosus peroxidase.

To understand the relationship between the structure and functions of the peroxidase of Arthromyces ramosus, a novel taxon of hyphomycete, and the evolutionary relationship of the A.ramosus peroxidase (ARP) with the other peroxidases, we isolated complementary and genomic DNA clones encoding ARP and characterized them. The sequence analyses of the ARP and cDNA coding for ARP showed that a mature ARP consists of 344 amino acids with a N-terminal pyroglutamic acid preceded by a signal peptide of 20 amino acid residues. The amino acid sequence of ARP was 99% identical to that of the peroxidase of Coprinus cinereus, a basidiomycete, and also had very high similarities (41-43% identity) to those of basidiomycetous lignin peroxidases, although we could find no lignin peroxidase activities for ARP when assayed with lignin model compounds. We could identified His184 and His56 as proximal and distal ligands to heme, respectively, and Arg52 as an essential Arg. Comparison of the sequences of complementary and genomic DNAs found that protein-encoding DNA is interrupted by 14 intervening sequences. The ARP cDNA was expressed in the yeast Saccharomyces cerevisiae under the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene, yielding 0.02 units/ml of a secreted active peroxidase.

Cloning, nucleotide sequence, and expression of an isovaleryl pepstatin-insensitive carboxyl proteinase gene from Pseudomonas sp. 101.

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.

Divalent metal ion requirements of a thermostable multimetal beta-galactosidase from Saccharopolyspora rectivirgula.

To understand the roles of metal ions on the catalytic properties and thermostability of the thermostable beta-galactosidase of Saccharopolyspora rectivirgula, a thermophilic actinomycete, we have investigated the binding kinetics and requirements of divalent metal ions by equilibrium dialysis, titration, and metal ion buffer techniques. We found that the monomeric multimetal enzyme (M(r) 136,977) had eight specific binding sites for divalent metal ions. These sites were classified as follows: a very tight (class I) site for Ca2+, three tight (class II) sites consisting of two Ca(2+)-specific sites (class IICa) and one Mn(2+)-specific site (class IIMn; Kd for Mn2+, 2.0 nM), and four loose (class III) sites for Mn2+ (Kd, 1.2 microM) and Mg2+ (Kd, 2 microM). Removal of metal ions bound to class II and III sites of the holoenzyme (Ca3Mn5 species; relative Vmax (Vrel), 100%) by a chelating resin at 4 degrees C yielded a less thermostable Ca1 species (Vrel, 1.7%) with a class I Ca2+ ion, removal of which by a chelating resin at 50 degrees C caused a complete irreversible inactivation of the enzyme. Titration studies revealed that stoichiometric binding of Mn2+ to a class IIMn site of the Ca1 species caused a 33-fold activation whereas binding of Ca2+ to class IICa sites had no effect on enzyme activity. Ca1 species could be also activated 8-fold by heating at 60 degrees C for 20 min, suggesting that the catalytically important class II Mn2+ plays important roles in maintaining the native structure essential for activity. Occupation of class III sites by Mg2+ or Mn2+ was of physiological importance to attain sufficient thermostability by which this extracellular beta-galactosidase remained active for a prolonged time at elevated temperatures as was observed during growth of S. rectivirgula.

Purification and characterization of a Bacillus sp. SAM1606 thermostable alpha-glucosidase with transglucosylation activity.

We purified a novel alpha-glucosidase to homogeneity from an Escherichia coli recombinant transformed with the alpha-glucosidase gene from thermophilic Bacillus sp. SAM1606. The enzyme existed as mono- and multimeric forms of a promoter protein with a relative molecular weight of 64,000 and isoelectric point of 4.6. We isolated a monomeric form of the enzyme and characterized it. The enzyme was unique among the known alpha-glucosidases in both broad substrate specificity and high thermostability. The enzyme hydrolysed a variety of O-alpha-D-glucopyranosides such as nigerose, maltose, isomaltose, sucrose, and trehalose efficiently. The molecular activity (k0) and the Michaelis constant (Km) values at 55 degrees C and pH 6.0 for sucrose were 54.6 s-1 and 5.3 mM, respectively. The optimum pH and temperature for hydrolysis were pH 5.5 and 75 degrees C, respectively. The enzyme exhibited a high transglucosylation activity: it reacted with 1.8 M sucrose at 60 degrees C for 70 h to yield oligosaccharides containing theanderose in a maximum yield of 35% (w/w). High thermostability of the enzyme (stable up to 65 degrees C at pH 7.2 for 10 min) permits the transglucosylation reaction at high temperatures, which would be beneficial for continuous production of oligosaccharides from sucrose.

Structure and expression of a gene coding for thermostable alpha-glucosidase with a broad substrate specificity from Bacillus sp. SAM1606.

We cloned an alpha-glucosidase gene from thermophilic Bacillus sp. SAM1606 to overexpress it in Escherichia coli transformants. Deletion of the 5'-noncoding region as well as expression of the alpha-glucosidase gene under the control of the icp promotor of the insecticidal crystal protein gene from Bacillus thuringiensis subsp. sotto enhanced the enzyme productivity to 23.5 U/ml, which was 12,000-fold higher than that obtained by the strain SAM1606. The open reading frame corresponding to the alpha-glucosidase encoded 587 amino acid residues including a residue coded by the initiation codon TTG, and the molecular mass of the alpha-glucosidase from N-terminal serine was calculated to be 68,886 Da. Sequence analysis revealed that the SAM1606 alpha-glucosidase belonged to the alpha-amylase family. The SAM1606 alpha-glucosidase showed extremely high sequence identity (62-65%) to the Bacillus cereus and Bacillus thermoglucosidasius oligo-1,6-glucosidases, which were 72% identical to each other. Sequence identity in the suggested active site regions were essentially the same (80-82%) among these three enzymes. However, the substrate specificity of the SAM1606 alpha-glucosidase was significantly different from those of the oligo-1,6-glucosidases. The thermostability of these three alpha-glucosidases could be correlated with the increase in the number of proline residues, whose occurrence was predicted at beta turns and coils in the enzymes.

Crystal structure of the fungal peroxidase from Arthromyces ramosus at 1.9 A resolution. Structural comparisons with the lignin and cytochrome c peroxidases.

The crystal structure of the peroxidase (donor: H2O2 oxidoreductase, EC 1.11.1.7) from the hyphomycete Arthromyces ramosus (ARP) has been determined by the multiple isomorphous replacement method and refined by the simulated annealing method to a crystallographic R-factor of 17.4% for the 19,191 reflections with F > 2 sigma F between 7.0 and 1.9 A resolution. The model includes residues 9 to 344, the heme group, two N-acetylglucosamine residues, two calcium ions and 246 water molecules. The root-mean-square deviation of bond lengths from the ideal values is 0.02 A. The mean coordinate error is estimated as 0.2 A. The electron density of the glycine-rich region of the amino-terminal eight residues was invisible. ARP has ten major and two short alpha-helices and a few short beta-strands. The overall tertiary structure of ARP is similar to that of yeast cytochrome c peroxidase (CCP) and is particularly similar to that of the lignin peroxidase (LiP) from Phanerochaete chrysosporium. Relative to CCP, ARP and LiP each have an extension of approximately 40 residues at the carboxy terminus. All eight cysteine residues in ARP form disulfide bonds (C12:C24, C23:C293, C43:C129 and C257:C322). Two calcium sites are inaccessible to solvent. The four disulfide bonds and two calcium sites, which are lacking in CCP, are conserved in ARP and LiP. The bond from Asn304C to Ala305N in ARP is the site sensitive to proteases. An Asx turn present in the Asn303 to Ala305 segment appears to orient the side-chain of Asn304 to outward from the molecule, rendering it easily trappable by pockets of proteases. The proximal heme ligand is His184 in helix F (distance of N epsilon 2 ... Fe, 2.10 A), and one of several water molecules in the distal pocket of the heme bridges the iron atom and the N epsilon 2 of His56. The orientation of the imidazole ring of the distal histidine residue relative to the heme group in ARP differs significantly from that in LiP. The access channel to the distal side of the heme of ARP is markedly wider along the heme plane than that of LiP. Many of the amino acid residues that comprise the entrance of this channel differ for ARP and LiP. This may account for the differences in substrate specificity.

Site-directed mutagenesis of Glu-141 and His-223 in Pseudomonas aeruginosa elastase: catalytic activity, processing, and protective activity of the elastase against Pseudomonas infection.

Both Pseudomonas aeruginosa elastase and Bacillus thermoproteolyticus thermolysin are zinc metalloproteases. On the basis of the high homology of the P. aeruginosa elastase with the Bacillus thermolysin, we hypothesized that Glu-141 and His-223 are the key residues for catalytic activity of the Pseudomonas elastase. To test this possibility, we replaced Glu-141 with Asp, Gln, and Gly and His-223 with Gly, Glu, and Leu by site-directed mutagenesis. These substitutions dramatically diminished the proteolytic activities of the mutant elastases when they were expressed in Escherichia coli cells. Although these mutant elastase precursors (proelastases) were produced, no appreciable processing was observed with these mutants. The possibility that autocatalysis is involved in both the processing and activation of elastase is discussed. Furthermore, by immunizing mice with vaccines made from these mutant elastase, we were able to obtain good protection against an intraperitoneal P. aeruginosa challenge.

Crystallization and preliminary X-ray diffraction studies of peroxidase from a fungus Arthromyces ramosus.

Peroxidase (donor: H2O2 oxidoreductase [EC 1.11.1.7]) was purified from the culture broth of the hyphomycete Arthromyces ramosus in the early log phase to show a single band on SDS-PAGE. The crystals of A. ramosus peroxidase (ARP) were formed by salting out with ammonium sulfate at room temperature and pH 7.5. The repeated seeding technique was employed to grow the crystals to the size large enough for X-ray diffraction study. The crystals were characterized as tetragonal, space group P4(2)2(1)2, with unit cell dimensions of a = b = 74.5 A, c = 117.6 A. The asymmetric unit contains one molecule of peroxidase. They diffract X-rays to at least 2.0 A resolution and are stable to X-rays.