Structure of a serine protease proteinase K from Tritirachium album limber at 0.98 A resolution

X-ray diffraction data at atomic resolution to 0.98 A with 136 380 observed unique reflections were collected using a high quality proteinase K crystals grown under microgravity conditions and cryocooled. The structure has been refined anisotropically with REFMAC and SHELX-97 with R-factors of 11.4 and 12.8%, and R(free)-factors of 12.4 and 13.5%, respectively. The refined model coordinates have an overall rms shifts of 0.23 A relative to the same structure determined at room temperature at 1.5 A resolution. Several regions of the main chain and the side chains, which were not observed earlier have been seen more clearly. For example, amino acid 207, which was reported earlier as Ser has been clearly identified as Asp. Furthermore, side-chain disorders of 8 of 279 residues in the polypeptide have been identified. Hydrogen atoms appear as significant peaks in the F(o) - F(c) difference electron density map accounting for an estimated 46% of all hydrogen atoms at 2sigma level. Furthermore, the carbon, nitrogen, and oxygen atoms can be differentiated clearly in the electron density maps. Hydrogen bonds are clearly identified in the serine protease catalytic triad (Ser-His-Asp). Furthermore, electron density is observed for an unusual, short hydrogen bond between aspartic acid and histidine in the catalytic triad. The short hydrogen bond, designated "catalytic hydrogen bond", occurs as part of an elaborate hydrogen bond network, involving Asp of the catalytic triad. Though unusual, these features seem to be conserved in other serine proteases. Finally there are clear electron density peaks for the hydrogen atoms associated with the Ogamma of Ser 224 and Ndelta1 of His 69.     

Isolation and characterization of the gene encoding a novel, thermostable serine proteinase from the mould Tritirachium album Limber

A number of proteinases are induced and secreted into the culture medium of Tritirachium album Limber when the nitrogen source is limited to exogenous proteins. We have constructed a cDNA library using the polyadenylated RNA isolated during the nutritional induction with bovine serum albumin. A full-length clone of a gene for a new proteinase (named proteinase R) was identified from this library. This clone contained sequences coding for the 108-amino-acid prepro-leader as well as for the 279-amino-acid mature proteinase. Proteinase R apparently belongs to the subtilisin group of serine proteases that contains disulphide bonds. Homology between proteinase R and proteinase K was found to be about 87% at the nucleotide as well as at the amino acid level. The Brookhaven Protein Data Base co-ordinate file of proteinase K was used as a template to study the proteinase R substitutions in three-dimensional space. The majority of the substitutions of proteinase R with respect to proteinase K were found to be on the exterior of the protein model.     

Nutritional regulation of proteinase production in the fungus, Tritirachium album

The fungus, Tritirachium album, produces a number of proteinases under proper conditions. We have studied the nutritional regulation mechanisms for proteinase production in the mold, i.e. the effects of carbon and nitrogen sources, and the influence of starvation. Proteinase production was induced when the nitrogen source was an exogenous protein or peptide, such as peptones or yeast extract. The production rate was affected by the amount of available inducing substrate. Inorganic nitrogen compounds, i.e., ammonium or nitrate salts, had a repressing effect on the production. Production was not induced if a detectable concentration of glucose or sucrose was present in the medium. Starvation did not trigger proteinase production. Journal of Industrial Microbiology & Biotechnology (2000) 24, 369–373. Received 20 January 1999/ Accepted in revised form 06 March 2000     

Proteinase K from Tritirachium album limber. I. Molecular mass and sequence around the active site serine residue

The molecular mass of proteinase K was determined by gel electrophoresis in the presence of sodium dodecyl sulfate and by active site labelling with diisopropyl fluorophosphate. Both methods indicate molecular masses in the range of 27 000-29 000 Da. These values differ significantly from that of 18 500 formerly determined by gel filtration (Ebeling et al. (1974) Eur. J. Biochem. 47, 91-97). Proteinase K was inactivated with [3H]diisopropyl fluorophosphate. Afterwards the labelled protein was reduced, S-carboxymethylated and digested with cyanogen bromide. The chain lengths of the isolated CNBr-fragments are indicative of a molecular mass of proteinase K of at least 28 000 Da. Two CNBr-fragments were sequenced. The radioactively labelled fragment contains 69 residues and the sequence around the labelled residues was found to be -Ile-Ser-Gly-Thr-SER-Met-Ala-Thr-Pro-. This sequence is typical for that around the active site residue of the subtilisins. From the determined sequences it is concluded that the fungal proteinase K is phylogenetically related to the bacterial subtilisins.     

Comparative study on proteinase R, T, and K from Tritirachiam album limber

Proteinase R and T purified from Tritirachiam album limber were characterized in comparison with proteinase K using circular dichroism (CD), enzyme activity, thermal melting, and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). CD analysis suggested that these three proteins possess some beta-sheet structure, with little alpha-helix except for proteinase R which showed about 14% alpha-helix. SDS-PAGE and gel filtration in 0.1% SDS indicated that proteinase T and K are resistant to SDS-induced unfolding similar to subtilisin. Thermal denaturation experiments showed the melting temperature for proteinase T to be 67 degrees and that for proteinase K to be 65 degrees in the absence of Ca2+, with higher melting temperatures in the presence of Ca2+. However, the enzyme activities of proteinase T and R were significantly lower than those of proteinase K.     

Cloning and sequencing of a gene encoding a novel extracellular neutral proteinase from Streptomyces sp. strain C5 and expression of the gene in Streptomyces lividans 1326.

The gene encoding a novel milk protein-hydrolyzing proteinase was cloned on a 6.56-kb SstI fragment from Streptomyces sp. strain C5 genomic DNA into Streptomyces lividans 1326 by using the plasmid vector pIJ702. The gene encoding the small neutral proteinase (snpA) was located within a 2.6-kb BamHI-SstI restriction fragment that was partially sequenced. The molecular mass of the deduced amino acid sequence of the mature protein was determined to be 15,740, which corresponds very closely with the relative molecular mass of the purified protein (15,500) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified neutral proteinase was determined, and the DNA encoding this sequence was found to be located within the sequenced DNA. The deduced amino acid sequence contains a conserved zinc binding site, although secondary ligand binding and active sites typical of thermolysinlike metalloproteinases are absent. The combination of its small size, deduced amino acid sequence, and substrate and inhibition profile indicate that snpA encodes a novel neutral proteinase.     

Structure and expression of the gene encoding cystatin D, a novel human cysteine proteinase inhibitor

A new member of the human cystatin multigene family has been cloned from a genomic library using a cystatin C cDNA probe. The complete nucleotide sequence of a 4.3-kilobase DNA segment, containing a complete gene with structure very similar to those of known Family 2 cystatin genes, was determined. The novel gene, called CST4, is composed of three exons and two introns. It contains the coding information for a protein of 142 amino acid residues, which has been tentatively called cystatin D. The deduced amino acid sequence includes a putative signal peptide and presents 51-55% identical residues with the sequences of either cystatin C or the secretory gland cystatins S, SN, or SA. The cystatin D sequence contains all regions of relevance for cysteine proteinase inhibitory activity and also the 4 cysteine residues that form disulfide bridges in the other members of cystatin Family 2. Northern blot analysis revealed that the cystatin D gene is expressed in parotid gland but not in seminal vesicle, prostate, epididymis, testis, ovary, placenta, thyroid, gastric corpus, small intestine, liver, or gall-bladder tissue. This tissue-restricted expression is in marked contrast with the wider distribution of all the other Family 2 cystatins, since cystatin C is expressed in all these tissues and the secretory gland cystatins are present in saliva, seminal plasma, and tears. Cystatin D, being the first described member of a third subfamily within the cystatin Family 2, thus appears to have a distinct function in the body in contrast to other cystatins.     

罗尼氏弧菌Vibrio shilonii BY新琼胶酶基因的克隆、序列分析及表达

【目的】从海洋来源的罗尼氏弧菌菌株BY中克隆得到一个具有琼胶酶活性的新基因,并对其进行重组表达。【方法】对实验室保藏的产琼胶酶菌株BY进行16S rRNA基因序列分析,并构建系统发育树。根据已报道的琼胶酶基因序列的同源性,设计简并引物,利用降落PCR (Touch-down PCR)及染色体步移技术扩增琼胶酶基因序列全长,对基因序列进行生物信息学分析。将目的基因插入pET22a(+)载体,转化大肠杆菌BL21(DE3),对重组酶进行表达,利用DNS法测定了重组酶的酶活,对该重组琼胶酶酶学性质进行研究。【结果】克隆得到一条新的琼胶酶基因,命名为Vibrio sp. BY (GenBank登录号：AIW39921.1),Vibrio sp. BY基因序列全长2 232 bp,编码744个氨基酸,理论分子量为85 kD,Vibrio sp. BY的氨基酸序列基因库中与已知的琼胶酶氨基酸序列Vibrio sp. EJY3的相似度为86%。发酵液琼胶酶酶活力为71.73 U/mL,证明表达的蛋白为琼胶酶。酶学性质研究表明重组琼胶酶的最适温度及pH分别为50 °C和7.0,并且具有较好的稳定性。【结论】利用染色体步移技术克隆得到一条新的琼胶酶基因,并在大肠杆菌BL21(DE3)中实现了重组表达,为琼胶酶的应用奠定了基础。     

Isolation and thermal stability studies of two novel serine proteinases from the fungus Tritirachium album Limber.

A number of serine proteinases are secreted into the culture medium when Tritirachium album Limber is supplied with protein as the only nitrogen source. From this population of proteinases, we have isolated two novel proteolytic enzymes, designated as proteinase R and T. We have compared the thermal stability of these two proteinases with that of subtilisin BPN' and proteinase K. Both of these proteinases were thermally stable in the absence of detergents in buffers of low (4.0) and high (10.0) pH. The thermal stability of proteinase T was not affected by the presence of 1.0% SDS either at pH 8.0 or 10.0 in contrast to proteinase R which became heat labile. At low pH, the presence of SDS was detrimental to the stability of all the proteinases.     

Cloning and expression of a novel protease gene encoding an extracellular neutral protease from Bacillus subtilis.

We have cloned from Bacillus subtilis a novel protease gene (nprB) encoding a neutral protease by using a shotgun cloning approach. The gene product was determined to have a molecular mass of 60 kDa. It has a typical signal peptide-like sequence at the N-terminal region. The expression of nprB can be stimulated by using a B. subtilis strain, WB30, carrying a sacU(h)h mutation. Expression of this protease gene results in production of a 37-kDa protease in the culture medium. The first five amino acid residues from the N terminus of the mature protease were determined to be Ala-Ala-Gly-Thr-Gly. This indicates that the protease is synthesized in a preproenzyme form. The purified protease has a pH optimum of around 6.6, and its activity can be inhibited by EDTA, 1,10-phenanthroline (a zinc-specific chelator), and dithiothreitol. It retained 65% of its activity after treatment at 65 degrees C for 20 min. Sequence comparison indicates that the mature form of this protease has 66% homology with the two thermostable neutral proteases from B. thermoproteolyticus and B. stearothermophilus. It also shares 65, 61, and 56% homology with the thermolabile neutral proteases from B. cereus, B. amyloliquefaciens, and B. subtilis, respectively. The zinc-binding site and the catalytic residues are all conserved among these proteases. Sequence homology extends into the "propeptide" region. The nprB gene was mapped between metC and glyB and was not required for growth or sporulation.     

Cloning and expression analysis of the gene encoding fibrinogen-like protein A,a novel regeneration-related protein from Apostichopus japonicus

Fibrinogen-like protein A (FGLA), a member of the fibrinogen-related protein superfamily, exists in different tissues of vertebrates and invertebrates. FGLA plays crucial roles including innate immune response, blood clotting and regeneration. In this study, the fibrinogen-like protein A (fglA) was cloned from Apostichopus japonicus using rapid amplification of cDNA ends PCR techniques. The cDNA sequence of fglA is 1,524 bp with a 849 bp open reading frame encoding a polypeptide of 282 amino acids, with an N-terminal signal peptide and a conserved C-terminal domain. Bioinformatic analysis revealed that the predicted molecular weight of the whole protein is 31.9 kDa and it has an isoelectric point of 5.64. In-situ hybridization demonstrated that fglA is widely distributed in body wall, intestines, longitudinal muscles and respiratory tree. The expression levels of fglA during different regeneration stages in the body wall, intestine and respiratory trees were analyzed by real-time PCR. The expression of fglA gradually increased within 1 h in body wall, and reached a plateau before decreasing to the basal level. This indicates that fglA is associated with the regeneration of Apostichopus japonicus.     

Cloning, sequencing and expression of the gene encoding the cell-envelope-associated proteinase from Lactobacillus paracasei subsp. paracasei NCDO 151.

The gene encoding the cell-envelope-associated proteinase of Lactobacillus paracasei subsp. paracasei NCDO 151 (formerly Lactobacillus casei NCDO 151) was cloned and sequenced. The gene was located on the chromosome and encoded a polypeptide of 1902 amino acids. The proteinase is N-terminally cleaved upon maturation. It shows extensive homology to the Lactococcus lactis subsp. cremoris Wg2 proteinase. Similar to the situation in Lactococcus, a maturation gene was found upstream of the proteinase gene. The cloned proteinase gene was expressed in Lactobacillus plantarum. However, no expression was observed when the gene was cloned in Lactococcus lactis.     

Cloning and expression of a gene encoding cyanidase from Pseudomonas stutzeri AK61

The gene coding for cyanidase, which catalyzes the hydrolysis of cyanide to formate and ammonia, was cloned from chromosomal DNA of Pseudomonas stutzeri AK61 into Escherichia coli. The cyanidase gene consisted of an open reading frame of 1004 bp, and it was predicted that cyanidase was composed of 334 amino acids with a calculated molecular mass of 37 518 Da. The amino acid sequence of cyanidase showed a 35.1% and 26.4% homology to aliphatic nitrilase from Rhodococcus rhodochrous K22 and cyanide hydratase from Fusarium lateritium, respectively. A unique cysteine residue of aliphatic nitrilase, which was suggested to play an essential role in the catalytic activity, was conserved in cyanidase. The active form of cyanidase was successfully expressed by a DNA clone containing the cyanidase gene in E.coli. Its productivity was approximately 230 times larger than that of P. stutzeri AK61. The characteristics of the expressed cyanidase, including optimum pH, optimum temperature, Michaelis constant (K _m) for cyanide and specific activity, were similar to those of the native enzyme from P. stutzeri AK61. Received: 24 October 1997 / Received last revision: 17 March 1998 / Accepted: 20 March 1998     

Cloning and expression of the Lactococcus lactis subsp. cremoris SK11 gene encoding an extracellular serine proteinase

The Lactococcus lactis subsp. cremoris SK11 plasmid-located prtP gene, encoding a cell-envelope-located proteinase (PrtP) that degrades alpha s1-, beta- and kappa-casein, was identified in a lambda EMBL3 gene library in Escherichia coli using immunological methods. The complete prtP gene could not be cloned in E. coli and L. lactis on high-copy-number plasmid vectors. However, using a low-copy-number vector, the complete prtP gene could be cloned in strains MG1363 and SK1128, proteinase-deficient derivatives of L. lactis subsp. lactis 712 and L. lactis subsp. cremoris SK11, respectively. The proteinase deficiency of these hosts was complemented to wild-type (wt) levels by the cloned SK11 prtP gene. The caseinolytic specificity of the proteinase specified by the cloned prtP gene was identical to that encoded by the wt proteinase plasmid, pSK111. The expression of recombinant plasmids containing 3' and 5' deletions of prtP was analyzed with specific attention directed towards the location of the gene products. In this way the expression signals of prtP were localized and overproduction was obtained in L. lactis subsp. lactis. Furthermore, a region at the C terminus of PrtP was identified which is involved in cell-envelope attachment in lactococci. A deletion derivative of prtP was constructed which specifies a C-terminally truncated proteinase that is well expressed and fully secreted into the medium, and still shows the same capacity to degrade alpha s1-, beta- and kappa-casein.     

Cloning, characterization, and expression of a novel gene encoding a reversible 4-hydroxybenzoate decarboxylase from Clostridium hydroxybenzoicum.

A novel gene, designated ohb1, which encodes the oxygen-sensitive and biotin-, ATP-, thiamin-, pyridoxal phosphate-, and metal-ion-independent, reversible 4-hydroxybenzoate decarboxylase (4-HOB-DC) from the obligate anaerobe Clostridium hydroxybenzoicum JW/Z-1(T) was sequenced (GenBank accession no. AF128880) and expressed. The 1,440-bp open reading frame (ORF) (ohb1) encodes 480 amino acids. Major properties of the heterologous enzyme (Ohb1) expressed in Escherichia coli DH5alpha were the same as those described for the native 4-HOB-DC (Z. He and J. Wiegel, J. Bacteriol. 178:3539-3543, 1996). The deduced amino acid sequence shows up to 57% identity and up to 74% similarity to hypothetical proteins deduced from ORFs in genomes from bacteria and archaea, suggesting a possible novel gene family.