Cloning,Sequencing, and Expression of the Gene Encoding 4-Hydroxy-4-methyl-2-oxoglutarate Aldolase from Pseudomonas ochraceae NGJ1

A DNA fragment that carried the gene (proA) encoding 4-hydroxy-4-methyl-2-oxoglutarate aldolase was cloned from the chromosomal DNA of Pseudomonas ochraceae NGJ1, and the coding region was assigned to the nucleotide sequence based on the N-terminal amino acid sequence of the enzyme purified from the organism. The proA gene was 684 bp long, corresponding to a protein of 227 amino acid residues with a calculated molecular mass of 24,067 Da. The genes encoding a putative transporter and a 4-oxalomesaconate hydratase were upstream, and a 3'-truncated gene encoding 2-pyrone-4,6-dicarboxylate lactonase was downstream from the proA gene in the same orientation on the DNA fragment. The proA gene product was overproduced in Escherichia coli and briefly purified to homogeneity from the crude extract by a two-step purification. The molecular and catalytic properties of the gene product were similar to those of the P. ochraceae enzyme.     

A new carboxylesterase from Brevibacterium linens IFO 12171 responsible for the conversion of 1,4-butanediol diacrylate to 4-hydroxybutyl acrylate: purification, characterization, gene cloning, and gene expression in Escherichia coli.

A carboxylesterase that is responsible for conversion of 1,4-butanediol diacrylate (BDA) to 4-hydroxybutyl acrylate (4HBA) was found in Brevibacterium lines IFO 12171, and purified to homogeneity. The purified enzyme was active toward a variety of diesters of ethylene glycol, 1,4-butanediol, and 1,6-hexanediol. The K(m) and kcat of the enzyme for BDA were 3.04 mM and 203,000 s-1, respectively. The reaction with the purified enzyme gave 98 mM 4HBA from 100 mM BDA for 60 min. The enzyme gene was cloned from the chromosomal DNA of the bacterium. The open reading frame encoding the enzyme was 1176 bp long, corresponding to a protein of 393 amino acid residues (molecular mass = 42,569 Da). The deduced amino acid sequence contained the tetra peptide motif sequence, STTK, and the serine residue was confirmed to be the catalytic center of BDA esterase by site-directed mutagenesis for several amino acid residues. The gene was expressed in Escherichia coli under the control of the lac promoter, and the gene product (a fusion protein with 6 amino acid residues from beta-galactosidase) showed the same catalytic properties as the enzyme from the parent strain.     

Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT.

4-Methyl-5-nitrocatechol (MNC) is an intermediate in the degradation of 2,4-dinitrotoluene by Burkholderia sp. strain DNT. In the presence of NADPH and oxygen, MNC monooxygenase catalyzes the removal of the nitro group from MNC to form 2-hydroxy-5-methylquinone. The gene (dntB) encoding MNC monooxygenase has been previously cloned and characterized. In order to examine the properties of MNC monooxygenase and to compare it with other enzymes, we sequenced the gene encoding the MNC monooxygenase and purified the enzyme from strain DNT. dntB was localized within a 2.2-kb ApaI DNA fragment. Sequence analysis of this fragment revealed an open reading frame of 1,644 bp with an N-terminal amino acid sequence identical to that of purified MNC monooxygenase from strain DNT. Comparison of the derived amino acid sequences with those of other genes showed that DntB contains the highly conserved ADP and flavin adenine dinucleotide (FAD) binding motifs characteristic of flavoprotein hydroxylases. MNC monooxygenase was purified to homogeneity from strain DNT by anion exchange and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of 60,200, which is consistent with the size determined from the gene sequence. The native molecular weight determined by gel filtration was 65,000, which indicates that the native enzyme is a monomer. It used either NADH or NADPH as electron donors, and NADPH was the preferred cofactor. The purified enzyme contained 1 mol of FAD per mol of protein, which is also consistent with the detection of an FAD binding motif in the amino acid sequence of DntB. MNC monooxygenase has a narrow substrate specificity. MNC and 4-nitrocatechol are good substrates whereas 3-methyl-4-nitrophenol, 3-methyl-4-nitrocatechol, 4-nitrophenol, 3-nitrophenol, and 4-chlorocatechol were not. These studies suggest that MNC monooxygenase is a flavoprotein that shares some properties with previously studied nitrophenol oxygenases.     

Molecular cloning, DNA sequence, and gene expression of the oxalyl-coenzyme A decarboxylase gene, oxc, from the bacterium Oxalobacter formigenes.

Oxalic acid, a highly toxic by-product of metabolism, is catabolized by a limited number of bacterial species by an activation-decarboxylation reaction which yields formate and CO2. oxc, the gene encoding the oxalic acid-degrading enzyme oxalyl-coenzyme A decarboxylase, was cloned from the bacterium Oxalobacter formigenes. The DNA sequence revealed a single open reading frame of 1,704 bp capable of encoding a 568-amino-acid protein with a molecular weight of 60,691. The identification of a presumed promoter region and a rho-independent termination sequence indicates that this gene is not part of a polycistronic operon. A PCR fragment encoding the open reading frame, when overexpressed in Escherichia coli, produced a product which cross-reacted antigenically with native enzyme on Western blots (immunoblots), appeared to form homodimers spontaneously, and exhibited enzymatic activity similar to that of the purified native enzyme.     

Cloning, sequence analysis, and expression of the bacteriophage T4 cd gene

The cd gene of bacteriophage T4, which encodes the enzyme deoxycytidylate deaminase, was isolated as a 1.9-kilobase DNA fragment and completely sequenced. The deduced amino acid sequence was found to be 193 residues long compared with 188 for the corresponding enzyme from bacteriophage T2. There were nine amino acid differences between the two enzymes in addition to a 5-residue insert near the carboxyl terminus of the T4 deaminase which was not present in the T2 deaminase. The cd-containing fragment also contained all of gene 31 (Nivinskas, R., and Black, L. W. (1988) Gene (Amst.) 73, 251-257) and thus precisely locates the two genes relative to one another within the T4 phage genomic map. Attempts to place the cd gene within a high expression vector have not been successful so far due to possible toxic effects of the gene product. However, placement of the gene within pUC18 resulted in a degree of expression which is about 10-20 times that found in T4-infected Escherichia coli. The enzyme was purified to homogeneity and found to possess properties similar to T2 phage deoxycytidylate deaminase.     

弗氏链霉菌丝氨酸蛋白酶基因的克隆及表达

从一株具有极强的降解羽毛能力的弗氏链霉菌菌株（Streptomyces fradiae var.k11）中纯化得到了一种丝氨酸蛋白酶SFP2。经蛋白测序,得到部分氨基酸序列,设计简并引物,PCR扩增得到部分基因序列,通过构建基因文库,获得了包括信号肽序列在内的完整的基因sfp2(EMBL收录号AJ784940),开放阅读框全长924bp,包括114bp的信号肽编码序列和810bp的酶原编码序列, 其中成熟蛋白编码基因长576bp,编码191个氨基酸,理论分子量为19.112kD。酶原编码基因和成熟蛋白编码基因均在大肠杆菌和枯草芽孢杆菌中得到了表达,酶原编码基因表达产物具有正常的生物学活性,证明了克隆基因的生物学功能。     

Deoxycytidylate hydroxymethylase gene of bacteriophage T4. Nucleotide sequence determination and over-expression of the gene

We describe two approaches to cloning and over-expressing gene 42 of bacteriophage T4, which encodes the early enzyme deoxycytidylate hydroxymethylase. In Bochum a library of sonicated fragments of wild-type phage DNA cloned into M13mp18 was screened with clones known to contain parts of gene 42. Two overlapping fragments, each of which contained one end of the gene, were cleaved at a HincII site and joined, to give a fragment containing the entire gene. In Corvallis a 1.8-kb fragment of cytosine-substituted DNA, believed to contain the entire gene, was cloned into pUC18 and shown to express the enzyme at low level. The cloned fragment bore an amber mutation in gene 42. From the DNA sequence of gene 42, the cloned gene was converted to the wild-type allele by site-directed mutagenesis. Both gene-42-containing fragments were cloned into the pT7 expression system and found to be substantially overexpressed. dCMP hydroxymethylase purified from one of the over-expressing strains had a turnover number similar to that of the enzyme isolated earlier from infected cells. In addition, the N-terminal 20 amino acid residues matched precisely the sequence predicted from the gene sequence. The amino acid sequence of gp42 bears considerable homology with that of thymidylate synthase of either host or T4 origin. The gene 42 nucleotide sequences of bacteriophages T2 and T6 were determined and found to code for amino acid sequences nearly identical to that of T4 gp42.     

枯草芽孢杆菌抗脯氨酸结构类似物突变株的筛选及突变株proBA基因的克隆和序列分析

利用亚硝基胍对枯草芽孢杆菌93151进行诱变处理,获得了耐NaCl浓度达14％的突变株,同时发现该突变株也是一个抗脯氨酸反馈抑制突变菌株,其胞内自由脯氨酸的含量随着盐浓度的提高显著增加,说明其对渗透压的耐受能力与胞内自由脯氨酸的含量紧密相关。利用PCR方法克隆突变株的proBA基因,得到一个约2.3kb的DNA片段,序列分析表明该片段含有一完整的proB基因和部分proA基因,与野生菌株的proB基因相比,突变株proB基因中有3个碱基发生了改变,其中一个碱基的变化（从起始密码子开始第781位由T→A）导致了一个氨基酸发生改变（Ser→Thr),另外两个碱基变化为沉默位点突变。将该proB基因转入大肠杆菌脯氨酸营养缺陷型菌株,能够与其功能互补。同时对部分proA基因序列分析发现,其与proB基因头尾重叠。在proA基因起始密码子上游第14个碱基处有一个类似于SD的序列,其所编码的氨基酸序列与枯草芽孢杆菌168的同源性为77％。     

Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes.

Two chitinases were purified from Rhizopus oligosporus, a filamentous fungus belonging to the class Zygomycetes, and designated chitinase I and chitinase II. Their N-terminal amino acid sequences were determined, and two synthetic oligonucleotide probes corresponding to these amino acid sequences were synthesized. Southern blot analyses of the total genomic DNA from R. oligosporus with these oligonucleotides as probes indicated that one of the two genes encoding these two chitinases was contained in a 2.9-kb EcoRI fragment and in a 3.6-kb HindIII fragment and that the other one was contained in a 2.9-kb EcoRI fragment and in a 11.5-kb HindIII fragment. Two DNA fragments were isolated from the phage bank of R. oligosporus genomic DNA with the synthetic oligonucleotides as probes. The restriction enzyme analyses of these fragments coincided with the Southern blot analyses described above and the amino acid sequences deduced from their nucleotide sequences contained those identical to the determined N-terminal amino acid sequences of the purified chitinases, indicating that each of these fragments contained a gene encoding chitinase (designated chi 1 and chi 2, encoding chitinase I and II, respectively). The deduced amino acid sequences of these two genes had domain structures similar to that of the published sequence of chitinase of Saccharomyces cerevisiae, except that they had an additional C-terminal domain. Furthermore, there were significant differences between the molecular weights experimentally determined with the two purified enzymes and those deduced from the nucleotide sequences for both genes. Analysis of the N- and C-terminal amino acid sequences of both chitinases and comparison of them with the amino acid sequences deduced from the nucleotide sequences revealed posttranslational processing not only at the N-terminal signal sequences but also at the C-terminal domains. It is concluded that these chitinases are synthesized with pre- and prosequences in addition to the mature enzyme sequences and that the prosequences are located at the C terminal.     

Purification, properties, and genetic location of Escherichia coli cytidine 5'-monophosphate N-acetylneuraminic acid synthetase

N-Acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-NeuAc synthetase) catalyzes the formation of cytidine monophosphate N-acetylneuraminic acid. We have purified CMP-NeuAc synthetase from an Escherichia coli O18:K1 cytoplasmic fraction to apparent homogeneity by ion exchange chromatography and affinity chromatography on CDP-ethanolamine linked to agarose. The enzyme has a specific activity of 2.1 mumol/mg/min and migrates as a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis. The enzyme has a requirement for Mg2+ or Mn2+ and exhibits optimal activity between pH 9.0 and 10. The apparent Michaelis constants for the CTP and NeuAc are 0.31 and 4 mM, respectively. The CTP analogues 5-mercuri-CTP and CTP-2',3'-dialdehyde are inhibitors. The purified CMP-N-acetylneuraminic acid synthetase has a molecular weight of approximately 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene encoding CMP-N-acetylneuraminic acid synthetase is located on a 3.3-kilobase HindIII fragment. The purified enzyme appears to be identical to the 50,000 Mr polypeptide encoded by this gene based on insertion mutations that result in the loss of detectable enzymatic activity. The amino-terminal sequence of the purified protein was used to locate the start codon for the CMP-NeuAc synthetase gene. Both the enzyme and the 50,000 Mr polypeptide have the same NH2-terminal amino acid sequence. Antibodies prepared to a peptide derived from the NH2-terminal amino acid sequence bind to purified CMP-NeuAc synthetase.     

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.     

Molecular cloning, overproduction and characterization of the Bacillus cereus IMP dehydrogenase

The gene of IMP dehydrogenase of Bacillus cereus ts-4, a temperature-sensitive mutant of B. cereus JCM 2152, was subcloned and its sequence was analyzed. A B. cereus ts-4 DNA fragment of 2,065 bp containing the entire impdh gene and flanking regions was sequenced. The fragment contained an open reading frame of 1,527 bp encoding 509 amino acids with a calculated molecular mass of 55,390 Da. The impdh sequence of JCM 2152 was also analyzed by TA cloning using PCR products amplified with primers from B. cereus ts-4 impdh gene. The gene amplified by PCR was expressed in Escherichia coli using a pET17 x b expression plasmid. The N-terminal amino acid sequence of the overproduced enzyme was identified as Met-Trp-Glu-Ser-Lys-Phe-Val-Lys-Glu-Gly-Leu-Thr-Phe-AspAsp-Val-Leu -Leu-Val- Pro. The overproduced enzyme was eluted at a molecular mass of about 225 kDa by gel filtration. The molecular mass of the subunit was estimated to be 56 kDa by SDS-PAGE. The overproduced enzyme was active against IMP, IDP, and ITP, and showed the highest activity at pH 9.5. These properties of the recombinant enzyme were almost identical to those of IMP dehydrogenase of B. cereus.     

Purification, characterization, gene cloning, sequencing, and overexpression of aminopeptidase N from Streptococcus thermophilus A.

The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys-7-amino-4-methylcoumarin at pH 7 and 37 degrees C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co2+, Zn2+, and Mn2+. Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on the N-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected. The N-terminal and short internal amino acid sequences of purified PepN were determined. By using synthetic primers and a battery of PCR techniques, the pepN gene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252. Amino acid sequence analysis of the pepN gene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. The pepN gene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the host Escherichia coli strain. The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme.     

Molecular cloning and expression of a thermostable xylose (glucose) isomerase gene, xylA, from Streptomyces chibaensis J-59

In the present study, the xylA gene encoding a thermostable xylose (glucose) isomerase was cloned from Streptomyces chibaensis J-59. The open reading frame of xylA (1167 bp) encoded a protein of 388 amino acids with a calculated molecular mass of about 43 kDa. The XylA showed high sequence homology (92% identity) with that of S. olivochromogenes. The xylose (glucose) isomerase was expressed in Escherichia coli and purified. The purified recombinant XylA had an apparent molecular mass of 45 kDa, which corresponds to the molecular mass calculated from the deduced amino acid and that of the purified wild-type enzyme. The N-terminal sequences (14 amino acid residues) of the purified protein revealed that the sequences were identical to that deduced from the DNA sequence of the xylA gene. The optimum temperature of the purified enzyme was 85 degrees C and the enzyme exhibited a high level of heat stability.     

Cloning and sequence analysis of the gene encoding 3-hexulose-6-phosphate synthase from the methylotrophic bacterium, Methylomonas aminofaciens 77a, and its expression in Escherichia coli

Abstract A DNA fragment of 550 bp was specifically amplified by PCR with primers based on the N-terminal sequence of the purified 3-hexulose-6-phosphate synthase from Methylomonas aminofaciens 77a and on that of a lysyl endopep(idase-derived peptide. Using this PCR product as a probe, a gene coding for 3-hexulose-6-phosphate synthase in M. aminofaciens 77a chromosomal DNA was cloned in Escherichia coli JM109. Sequencing analysis revealed that the gene encoding 3-hexulose-6-phosphate synthase contained a 624-bp open reading frame, encoding a protein composed of 208 amino acid residues with a calculated relative molecular mass of 21 224.     

Triphenylmethane reductase from Citrobacter sp. strain KCTC 18061P: purification, characterization, gene cloning, and overexpression of a functional protein in Escherichia coli

We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60 degrees C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.     

Analysis of the Escherichia coli proBA locus by DNA and protein sequencing

A 2.9 kb DNA fragment carrying the Escherichia coli proBA region, which encodes the first two enzymes of the proline biosynthetic pathway, was subcloned onto an expression plasmid carrying both the bacteriophage lambda PL promoter (lambda PL) and the lambda gene encoding a thermolabile cI repressor protein (cI857). Derepression of the lambda PL promoter by thermal inactivation of the cI857 repressor protein resulted in the simultaneous overproduction of the proB (gamma-glutamyl kinase) and proA (gamma-glutamyl phosphate reductase) gene products. Nucleotide sequence analysis of the proBA locus allowed gene assignments consistent with the NH2 and COOH-terminal analyses and amino acid compositions of homogeneous preparations of the proB and proA proteins. The contiguous nature of the proB and proA genes suggests that the two genes constitute an operon in which proB precedes proA.     

Biochemical and genetic characterization of 2-carboxybenzaldehyde dehydrogenase, an enzyme involved in phenanthrene degradation by Nocardioides sp. strain KP7.

2-Carboxybenzaldehyde dehydrogenase from the phenanthrene-degrading bacterium Nocardioides sp. strain KP7 was purified and characterized. The purified enzyme had a molecular mass of 53 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 205 kDa by gel filtration chromatography. Thus, the homotetramer of the 53-kDa subunit constituted an active enzyme. The apparent Km and kcat values of this enzyme for 2-carboxybenzaldehyde were 100 microM and 39 s(-1), respectively, and those for NAD+ were 83 microM and 32 s(-1), respectively. The structural gene for this enzyme was cloned and sequenced. The length of the gene was 1,455 bp. The nucleotide sequence of the 10,279 bp of DNA around the gene for 2-carboxybenzaldehyde dehydrogenase was also determined, and seven open reading frames were found in this DNA region. These were the genes for 1-hydroxy-2-naphthoate dioxygenase (phdI) and trans-2'-carboxybenzalpyruvate aldolase (phdJ), orf1, the gene for 2-carboxybenzaldehyde dehydrogenase (phdK), orf2/orf3, and orf4. The amino acid sequence of the orf1 product was similar to that of the aromatic hydrocarbon transporter gene (pcaK) in Pseudomonas putida PRS2000. The amino acid sequence of the orf4 product revealed a similarity to cytochrome P-450 proteins. The region between phdK and orf4 encoded orf2 and orf3 on different strands. The amino acid sequences of the orf2 and orf3 products exhibited no significant similarity to the reported sequences in protein databases.