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1.
Prolyl endopeptidase from Flavobacterium meningosepticum: cloning and sequencing of the enzyme gene. 总被引:2,自引:0,他引:2
T Yoshimoto A Kanatani T Shimoda T Inaoka T Kokubo D Tsuru 《Journal of biochemistry》1991,110(6):873-878
The prolyl endopeptidase [EC 3.4.21.26] gene of Flavobacterium meningosepticum was cloned in Escherichia coli with the aid of an oligonucleotide probe which was prepared based on the amino acid sequence. The hybrid plasmid, pFPEP1, with a 3.5 kbp insert at the HincII site of pUC19 containing the enzyme gene, was subcloned into pUC19 to construct plasmid pFPEP3. The whole nucleotide sequence of an inserted HincII-BamHI fragment of plasmid pFPEP3 was determined by the dideoxy chain-terminating method. The purified prolyl endopeptidase was labeled with tritium DFP, and the sequence surrounding the reactive serine residue was found to be Ala (551)-Leu-Ser-Gly-Arg-*Ser-Asn(557). Ser-556 was identified as a reactive serine residue. The enzyme consists of 705 amino acid residues as deduced from the nucleotide sequence and has a molecular weight of 78,705, which coincides well with the value estimated by ultra centrifugal analysis. The amino acid sequence was 38.2% homologous to that of the porcine brain prolyl endopeptidase [Rennex et al. (1991) Biochemistry 30, 2195-2203] and 24.5% homologous to E. coli protease II, which has substrate specificity for basic amino acids [Kanatani et al. (1991) J. Biochem. 110, 315-320]. 相似文献
2.
cDNA cloning of porcine brain prolyl endopeptidase and identification of the active-site seryl residue 总被引:8,自引:0,他引:8
Prolyl endopeptidase is a cytoplasmic serine protease. The enzyme was purified from porcine kidney, and oligonucleotides based on peptide sequences from this protein were used to isolate a cDNA clone from a porcine brain library. This clone contained the complete coding sequence of prolyl endopeptidase and encoded a polypeptide with a molecular mass of 80,751 Da. The deduced amino acid sequence of prolyl endopeptidase showed no sequence homology with other known serine proteases. [3H]Diisopropyl fluorophosphate was used to identify the active-site serine of prolyl endopeptidase. One labeled peptide was isolated and sequenced. The sequence surrounding the active-site serine was Asn-Gly-Gly-Ser-Asn-Gly-Gly. This sequence is different from the active-site sequences of other known serine proteases. This difference and the lack of overall homology with the known families of serine proteases suggest that prolyl endopeptidase represents a new type of serine protease. 相似文献
3.
S Chevallier P Goeltz P Thibault D Banville J Gagnon 《The Journal of biological chemistry》1992,267(12):8192-8199
A prolyl endopeptidase was purified from Flavobacterium meningosepticum. It was digested with trypsin. Two oligonucleotides, based on tryptic peptide sequences and used in PCR experiments, amplified a 300-base pair (bp) fragment. A 2.4-kilobase EcoRI fragment that hybridized to the 300-bp probe was cloned in lambda ZAP and sequenced from both strands. It contains a reading frame of 2115 bp, encoding the complete protein sequence of 705 amino acids. Ion-spray mass spectrometry experiments demonstrated the presence of an NH2-terminal signal peptide: the periplasmic mature protease is 685 residues in length for a molecular mass of 76784 Da. The prolyl endopeptidase showed no general sequence homology with known protein sequences except with that of porcine brain prolyl endopeptidase. In order to identify the active-site serine, the prolyl endopeptidase was labeled with [3H]diisopropyl fluorophosphate. One labeled peptide was purified and sequenced. The active-site serine was located in position 536 within the sequence GRSNGG. This sequence is different from the active-site sequence of the trypsin (GDSGGP) and subtilisin (GTSMAS) families. 相似文献
4.
Fatty acid synthetase, malic enzyme and other NADP+ binding dehydrogenases have similar antigenic determinant(s) at the NADPH binding domain 总被引:2,自引:0,他引:2
The sequence of tryptic and chymotryptic peptides from cytosolic and mitochondrial rabbit liver serine hydroxymethyltransferase are compared to the proposed sequence of a protein coded for by the glyA gene of Escherichia coli. The E. coli glyA gene is believed to code for serine hydroxymethyltransferase. Extensive sequence homology between these peptides were found for the proposed E. coli enzyme in the aminoterminal two-thirds of the molecule. All three proteins have identical sequences from residue 222-231. This sequence is known to contain the lysyl residue which forms a Schiff's base with pyridoxal-P in the two rabbit liver enzymes. These results support the interpretation that the proposed sequence of E. coli serine hydroxymethyltransferase is correct. The data also show that cytosolic and mitochondrial serine hydroxymethyltransferase are homologous proteins. 相似文献
5.
从土样分离到一株产生具有立体选择性酯水解酶的恶臭假单孢菌(Pseudomonas putida NH33)。构建P.putida NH33的基因组文库,并在E.coli中进行酯酶活性筛选,得到一个含有4.7kb插入片段的阳性克隆。对这个克隆的DNA片段进行序列分析,表明它含有一个1142个碱基的开放阅读框,为编码381个氨基酸的酯酶。推测的酯酶氨基酸序列与其它丝氨酸酯水解酶具有共同的保守基序GXSXG。把该蛋白在E.coli BL21(DE3)中进行表达,并用金属亲和层析纯化至单一条带。利用纯化的酶水解2-芳基丙酸乙酯制备2-芳基丙酸的S型异构体,产物的光学纯度eep>99%,说明此酶可8用于手性药物的合成。该酯酶是一个新酶,其基因序列已递交GenBank,登记号为AY896293。 相似文献
6.
Cloning, nucleotide sequence, and expression of the Pasteurella haemolytica A1 glycoprotease gene. 总被引:11,自引:0,他引:11
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Pasteurella haemolytica serotype A1 secretes a glycoprotease which is specific for O-sialoglycoproteins such as glycophorin A. The gene encoding the glycoprotease enzyme has been cloned in the recombinant plasmid pH1, and its nucleotide sequence has been determined. The gene (designated gcp) codes for a protein of 35.2 kDa, and an active enzyme protein of this molecular mass can be observed in Escherichia coli clones carrying pPH1. In vivo labeling of plasmid-encoded proteins in E. coli maxicells demonstrated the expression of a 35-kDa protein from pPH1. The amino-terminal sequence of the heterologously expressed protein corresponds to that predicted from the nucleotide sequence. The glycoprotease is a neutral metalloprotease, and the predicted amino acid sequence of the glycoprotease contains a putative zinc-binding site. The gene shows no significant homology with the genes for other proteases of procaryotic or eucaryotic origin. However, there is substantial homology between gcp and an E. coli gene, orfX, whose product is believed to function in the regulation of macromolecule biosynthesis. 相似文献
7.
Specific excretion of Serratia marcescens protease through the outer membrane of Escherichia coli. 总被引:34,自引:22,他引:12
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A DNA fragment of Serratia marcescens directing an extracellular serine protease (Mr, 41,000) was cloned in Escherichia coli. The cloned fragment caused specific excretion of the protease into the extracellular medium through the outer membrane of E. coli host cells in parallel with their growth. No excretion of the periplasmic enzymes of host cells occurred. The cloned fragment contained a single open reading frame of 3,135 base pairs coding a protein of 1,045 amino acids (Mr 112,000). Comparison of the 5' nucleotide sequence with the N-terminal amino acid sequence of the protease indicated the presence of a typical signal sequence. The C-terminal amino acid of the enzyme was found at position 408, as deduced from the nucleotide sequence. Artificial frameshift mutations introduced into the coding sequence for the assumed distal polypeptide after the C terminus of the protease caused complete loss of the enzyme production. It was concluded that the Serratia serine protease is produced as a 112-kilodalton proenzyme and that its N-terminal signal peptide and a large C-terminal part are processed to cause excretion of the mature protease through the outer membrane of E. coli cells. 相似文献
8.
Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein. 总被引:18,自引:0,他引:18
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The sequence of a 1,693-base-pair plasmid DNA fragment from Flavobacterium sp. strain ATCC 27551 containing the parathion hydrolase gene (opd) was determined. Within this sequence, there is only one open reading frame large enough to encode the 35,000-dalton membrane-associated hydrolase protein purified from Flavobacterium extracts. Amino-terminal sequence analysis of the purified Flavobacterium hydrolase demonstrated that serine is the amino-terminal residue of the hydrolase protein. The amino-terminal serine corresponds to a TCG codon located 87 base pairs downstream of the presumptive ATG initiation codon in the nucleotide sequence. The amino acid composition of the purified protein agrees well with that predicted from the nucleotide sequence, using serine as the amino-terminal residue. These data suggest that the parathion hydrolase protein is processed at its amino terminus in Flavobacterium sp. Construction in Escherichia coli of a lacZ-opd gene fusion in which the first 33 amino-terminal residues of opd were replaced by the first 5 residues of lacZ resulted in the production of an active hydrolase identical in molecular mass to the hydrolase isolated from Flavobacterium sp. E. coli cells containing the lacZ-opd fusion showed higher levels of hydrolase activity than did cells containing the parent plasmid. 相似文献
9.
枯草芽孢杆菌碱性蛋白酶基因的克隆和表达 总被引:2,自引:0,他引:2
目的:获得碱性蛋白酶基因。方法:用PCR的方法从枯草芽孢杆菌A-109中扩增碱性蛋白酶基因(apr),并进行测序分析,构建表达载体,最后转化大肠杆菌BL21,SDS-聚丙烯酰胺凝胶电泳检测该基因的表达情况。结果:apr基因片段含1092个碱基对。该基因片段核苷酸序列与Bacillus amyloliquefaciens subtilisin DFE precursor有99%的同源性,对应的氨基酸序列与Bacillussp.DJ-4有99%的同源性。apr基因在大肠杆菌BL21中获得表达,并表现出蛋白酶活性。结论:获得了具有活性的新的碱性蛋白酶基因。 相似文献
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12.
Molecular cloning and nucleotide sequence of the beta-lytic protease gene from Achromobacter lyticus. 总被引:3,自引:2,他引:1
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Two bacteriolytic enzymes secreted by Achromobacter lyticus M497-1 were purified and identified as being very similar (considering their amino acid composition and N-terminal sequence) to alpha- and beta-lytic proteases from Lysobacter enzymogenes. A 1.8-kb EcoRI fragment containing the structural gene for beta-lytic protease was cloned from A. lyticus chromosomal DNA. The protein sequence deduced from the nucleotide sequence was identical to the known sequence of beta-lytic protease, except for six residues. The nucleotide sequence revealed that the mature enzyme is composed of 179 amino acid residues with an additional 195 amino acids at the amino-terminal end of the enzyme, which includes the signal peptide, thus indicating that the enzyme is synthesized as a precursor protein. 相似文献
13.
Nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II and analyses of the deduced protein sequence 总被引:2,自引:0,他引:2
Jung-Kee Lee Moon-Hee Sung Ki-Hong Yoon Ju-Hyun Yu Tae-Kwang Oh 《FEMS microbiology letters》1994,119(1-2):137-145
Abstract The complete nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II (EIIMan ) was determined. The gene consisted of 2052 base pairs encoding a protein of 683 amino acid residues; the molecular mass of the protein subunit was calculated to be 72570 Da. The N-terminal hydrophilic domain of EIIMan showed 39.7% homology with a C-terminal hydrophilic domain of Escherichia coli glucose-specific enzyme II (EIIGlc ). Similar homology was shown between the C-terminal sequence of EIIMan and the E. coli glucose-specific enzyme III (EIIIGlc ), or the EIII-like domain of Streptococcus mutans sucrose-specific enzyme II. Sequence comparison with other EIIs showed that EIIMan contained residues His-602 and Cys-28 which were homologous to the potential phosphorylation sites of EIIIGlc , or EIII-like domains, and hydrophilic domains (IIB) of several EIIs, respectively. 相似文献
14.
The nucleotide sequence of gene 21 of bacteriophage T4 coding for the prohead protease 总被引:3,自引:0,他引:3
We have sequenced gene 21 coding for the bacteriophage T4 prohead protease. The sequence codes for a protein of 212 amino acids (aa) with an Mr of 23,251. A second possible in-frame initiation site was also found which would code for an Mr 18,440 protein. Evidence is presented that this second site is used in vivo. The only striking homology of gp21 to other proteins is with the serine proteases. The protein is homologous to a short aa sequence around the active site, but has a His where the active site Ser is normally found. However, mutation of this His to Ser gave a functional protein that could not be inhibited by serine protease inhibitors. We have located three sites in the gene that give rise to temperature-sensitive mutations. One of these is towards the N-terminus of the gene, the other two flank the region that shows homology with serine proteases. Attempts to overproduce the protein in Escherichia coli failed due to the extreme lability of the enzyme. A frame-shift mutation in the gene was therefore constructed which allowed the synthesis of large amounts of a stable N-terminal fragment of the protein. 相似文献
15.
The prolyl endopeptidase from pig brain was purified to homogeneity according to SDS-gel electrophoresis and visualization with the silver staining procedure. The molecular weight of prolyl endopeptidase was estimated as 70 kDa, and the isoelectric point as 4.9. The molecular properties of prolyl endopeptidase from pig brain are therefore similar to those of prolyl endopeptidases from other mammalian tissues. Diisopropylfluorophosphate, diethylpyrocarbonate and p-chloromercuribenzoic acid are strong irreversible inhibitors of prolyl endopeptidase from pig brain. We showed that diisopropylfluorophosphate und diethylpyrocarbonate act as competitive inhibitors with respect to substrate. Therefore it is assumed that at least one serine and one histidine residue are located at the active site of this enzyme. This result supports the assumption that the prolyl endopeptidase from pig brain is a typical serine protease. Substance P, thyreoliberin, beta-casomorphin-5 and morphiceptin are hydrolysed by prolyl endopeptidase in vitro. 相似文献
16.
An SY Ok M Kim JY Jang MS Cho YS Choi YL Kim CH Lee YC 《Indian journal of biochemistry & biophysics》2004,41(4):141-147
A gene, isp-B, encoding an intracellular serine protease from a newly isolated Bacillus sp. WRD-2 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 960 bp encoding a polypeptide comprised of 319 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other intracellular serine proteases, including active sites, Ser, His and Asp, as well as no signal sequence. The predicted amino acid sequence showed more than 60% homology with the intracellular serine proteases from Bacillus species. When expressed in E. coli, the recombinant enzyme (rISP-B) was overproduced in the cytoplasm as soluble and active form. The purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, EDTA and antipain. The enzyme showed maximum activity at pH 8.0 and 45 degrees C. It was stable atpH from 7.5 to 11.0 and below 50 degrees C. 相似文献
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18.
Cloning and sequencing of the 7 alpha-hydroxysteroid dehydrogenase gene from Escherichia coli HB101 and characterization of the expressed enzyme. 总被引:7,自引:6,他引:1
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T Yoshimoto H Higashi A Kanatani X S Lin H Nagai H Oyama K Kurazono D Tsuru 《Journal of bacteriology》1991,173(7):2173-2179
The 7 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) gene from Escherichia coli HB101 was cloned and expressed in E. coli DH1. The hybrid plasmid pSD1, with a 2.8-kbp insert of chromosomal DNA at the BamHI site of pBR322, was subcloned into pUC19 to construct plasmid pSD3. The entire nucleotide sequence of an inserted PstI-BamHI fragment of plasmid pSD3 was determined by the dideoxy chain-termination method. Within this sequence, the mature enzyme protein-encoding sequence was found to start at a GTG initiation codon and to comprise 765 bp, as judged by comparison with the protein sequence. The deduced amino acid sequence of the enzyme indicated that the molecular weight is 26,778. The transformant of E. coli DH1 harboring pSD3 with a 1.8-kbp fragment showed about 200-fold-higher enzyme activity than the host. The enzyme was purified by a single chromatography step on DEAE-Toyopearl and obtained as crystals, with an activity yield of 39%. The purified enzyme was homogeneous, as judged by sodium dodecyl sulfate gel electrophoresis. The enzyme was most active at pH 8.5 and stable between pH 8 and 9. The enzyme was NAD+ dependent and had a pI of 4.3. The molecular mass was estimated to be 120 kDa by the gel filtration method and 28 kDa by electrophoresis, indicating that the enzyme exists in a tetrameric form. 相似文献
19.
N G Seidah R Routhier M Caron M Chrétien S Demassieux R Boucher J Genest 《Canadian journal of biochemistry》1978,56(9):920-925
In this paper, we present the amino-terminal sequence of rat tonin, an endopeptidase responsible for the conversion of angiotensinogen, the tetradecapeptide renin substrate, or angiotensin I to angiotensin II. It is shown that isoleucine and proline occupy the amino- and carboxy-terminal residues respectively. The N-terminal sequence analysis permitted the identification of 34 out of the first 40 residues of the single polypeptide chain composed of 272 amino acids. These results showed an extensive homology with the sequence of many serine proteases of the trypsin-chymotrypsin family. This information, coupled with the slow inhibition of tonin by diisopropylfluorophosphate, classified this enzyme as a selective endopeptidase of the active serine protease family. 相似文献
20.
Nucleotide sequence of the Vibrio alginolyticus calcium-dependent, detergent-resistant alkaline serine exoprotease A 总被引:1,自引:0,他引:1
The nucleotide sequence of the Vibrio alginolyticus alkaline serine exoprotease A (ProA) gene cloned in Escherichia coli was determined. The exoprotease A gene (proA) consisted of 1602 bp which encoded a protein of 534 amino acids (aa) with an Mr of 55,900. The region upstream from the gene was characterized by a putative promoter consensus region (-10 -35), a ribosome-binding site and ATG start codon. The proA gene encodes a typical 21-aa N-terminal signal sequence which, when fused to alkaline phosphatase by means of transposon TnphoA, was able to mediate transport of the alkaline phosphatase to the periplasm in E. coli. Deletions of up to 106 aa from the C terminus of ProA did not result in the loss of extracellular protease activity. Additional V. alginolyticus genes were not involved in the secretion into the medium of the cloned ProA in E. coli. The amino acid sequence of ProA showed low overall homology to a Serratia marcescens serine exoprotease but significant homology was detected with other subtilisin family exoproteases. The fungal proteinase K, another sodium dodecyl sulfate-resistant protease, had 44% aa homology with ProA. 相似文献