Cloning, sequencing, and expression of the Zymomonas mobilis phosphoglycerate mutase gene (pgm) in Escherichia coli.

Phosphoglycerate mutase is an essential glycolytic enzyme for Zymomonas mobilis, catalyzing the reversible interconversion of 3-phosphoglycerate and 2-phosphoglycerate. The pgm gene encoding this enzyme was cloned on a 5.2-kbp DNA fragment and expressed in Escherichia coli. Recombinants were identified by using antibodies directed against purified Z. mobilis phosphoglycerate mutase. The pgm gene contains a canonical ribosome-binding site, a biased pattern of codon usage, a long upstream untranslated region, and four promoters which share sequence homology. Interestingly, adhA and a D-specific 2-hydroxyacid dehydrogenase were found on the same DNA fragment and appear to form a cluster of genes which function in central metabolism. The translated sequence for Z. mobilis pgm was in full agreement with the 40 N-terminal amino acid residues determined by protein sequencing. The primary structure of the translated sequence is highly conserved (52 to 60% identity with other phosphoglycerate mutases) and also shares extensive homology with bisphosphoglycerate mutases (51 to 59% identity). Since Southern blots indicated the presence of only a single copy of pgm in the Z. mobilis chromosome, it is likely that the cloned pgm gene functions to provide both activities. Z. mobilis phosphoglycerate mutase is unusual in that it lacks the flexible tail and lysines at the carboxy terminus which are present in the enzyme isolated from all other organisms examined.     

Cloning of the Zymomonas mobilis structural gene encoding alcohol dehydrogenase I (adhA): sequence comparison and expression in Escherichia coli.

Zymomonas mobilis ferments sugars to produce ethanol with two biochemically distinct isoenzymes of alcohol dehydrogenase. The adhA gene encoding alcohol dehydrogenase I has now been sequenced and compared with the adhB gene, which encodes the second isoenzyme. The deduced amino acid sequences for these gene products exhibited no apparent homology. Alcohol dehydrogenase I contained 337 amino acids, with a subunit molecular weight of 36,096. Based on comparisons of primary amino acid sequences, this enzyme belongs to the family of zinc alcohol dehydrogenases which have been described primarily in eucaryotes. Nearly all of the 22 strictly conserved amino acids in this group were also conserved in Z. mobilis alcohol dehydrogenase I. Alcohol dehydrogenase I is an abundant protein, although adhA lacked many of the features previously reported in four other highly expressed genes from Z. mobilis. Codon usage in adhA is not highly biased and includes many codons which were unused by pdc, adhB, gap, and pgk. The ribosomal binding region of adhA lacked the canonical Shine-Dalgarno sequence found in the other highly expressed genes from Z. mobilis. Although these features may facilitate the expression of high enzyme levels, they do not appear to be essential for the expression of Z. mobilis adhA.     

Phosphoglycerate kinase gene from Zymomonas mobilis: cloning, sequencing, and localization within the gap operon.

The Zymomonas mobilis gene encoding phosphoglycerate kinase (EC 2.7.3.2), pgk, has been cloned into Escherichia coli and sequenced. It consists of 336 amino acids, including the N-terminal methionine, with a molecular weight of 41,384. This promoterless gene is located 225 base pairs downstream from the gap gene and is part of the gap operon. Previous studies have shown that the specific activities of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase do not change coordinately in Z. mobilis, although the two enzymes appear to be under the control of a common promoter. The translated amino acid sequence for the Z. mobilis phosphoglycerate kinase is less conserved than those of eucaryotic genes. A comparison of known sequences for phosphoglycerate kinase revealed a high degree of conservation of structure with 102 amino acid positions being retained by all. In general, the amino acid positions at the boundaries of beta-sheet and alpha-helical regions and those connecting these regions were more highly conserved than the amino acid positions within regions of secondary structure.     

Cloning, sequencing, and characterization of the principal acid phosphatase, the phoC+ product, from Zymomonas mobilis.

The Zymomonas mobilis gene encoding acid phosphatase, phoC, has been cloned and sequenced. The gene spans 792 base pairs and encodes an Mr 28,988 polypeptide. This protein was identified as the principal acid phosphatase activity in Z. mobilis by using zymograms and was more active with magnesium ions than with zinc ions. Its promoter region was similar to the -35 "pho box" region of the Escherichia coli pho genes as well as the regulatory sequences for Saccharomyces cerevisiae acid phosphatase (PHO5). A comparison of the gene structure of phoC with that of highly expressed Z. mobilis genes revealed that promoters for all genes were similar in degree of conservation of spacing and identity with the proposed Z. mobilis consensus sequence in the -10 region. The phoC gene contained a 5' transcribed terminus which was AT rich, a weak ribosome-binding site, and less biased codon usage than the highly expressed Z. mobilis genes.     

Cloning and expression of the enolase gene from Desulfovibrio vulgaris (Miyazaki F)

The gene encoding an enolase from Desulfovibrio vulgaris (Miyazaki F) was cloned and overexpressed in Escherichia coli. A 2.1-kb DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with PstI and BamHI, contained an enolase gene (eno) and part of the methylenetetrahydrofolate dehydrogenase gene (folD). The nucleotide sequence of eno indicates that the protein monomer is composed of 434 amino acids. An expression system for eno under control of the T7 promoter was constructed in E. coli. The purified His-tagged enolase formed a homooctamer and was active in the formation of phosphoenolpyruvate (PEP) as well as in the reverse reaction, the formation of D-(+)-2-phosphoglyceric acid (2-PGA). The pH dependence and kinetic properties of the recombinant enolase from the sulfate-reducing bacterium were also studied. The amounts of eno mRNA when the bacterium was grown on glycerol or glucose were compared to that when D. vulgaris was grown on lactate.     

Nucleotide sequence of Escherichia coli pyrG encoding CTP synthetase

The amino acid sequence of Escherichia coli CTP synthetase was derived from the nucleotide sequence of pyrG. The derived amino acid sequence, confirmed at the N terminus by protein sequencing, predicts a subunit of 544 amino acids having a calculated Mr of 60,300 after removal of the initiator methionine. A glutamine amide transfer domain was identified which extends from approximately amino acid residue 300 to the C terminus of the molecule. The CTP synthetase glutamine amide transfer domain contains three conserved regions similar to those in GMP synthetase, anthranilate synthase, p-aminobenzoate synthase, and carbamoyl-P synthetase. The CTP synthetase structure supports a model for gene fusion of a trpG-related glutamine amide transfer domain to a primitive NH3-dependent CTP synthetase. The major 5' end of pyrG mRNA was localized to a position approximately 48 base pairs upstream of the translation initiation codon. Translation of the gene eno, encoding enolase, is initiated 89 base pairs downstream of pyrG. The pyrG-eno junction is characterized by multiple mRNA species which are ascribed to monocistronic pyrG and/or eno mRNAs and a pyrG eno polycistronic mRNA.     

Cloning and sequencing of the sacA gene: characterization of a sucrase from Zymomonas mobilis.

The Zymomonas mobilis gene (sacA) encoding a protein with sucrase activity has been cloned in Escherichia coli and its nucleotide sequence has been determined. Potential ribosome-binding site and promoter sequences were identified in the region upstream of the gene which were homologous to E. coli and Z. mobilis consensus sequences. Extracts from E. coli cells, containing the sacA gene, displayed a sucrose-hydrolyzing activity. However, no transfructosylation activity (exchange reaction or levan formation) could be detected. This sucrase activity was different from that observed with the purified extracellular protein B46 from Z. mobilis. These two proteins showed different electrophoretic mobilities and molecular masses and shared no immunological similarity. Thus, the product of sacA (a polypeptide of 58.4-kDa molecular mass) is a new sucrase from Z. mobilis. The amino acid sequence, deduced from the nucleotide sequence of sacA, showed strong homologies with the sucrases from Bacillus subtilis, Salmonella typhimurium, and Vibrio alginolyticus.     

The hyperthermophilic bacterium Thermotoga neapolitana possesses two isozymes of the 3-phosphoglycerate kinase/triosephosphate isomerase fusion protein

Abstract The phosphoglycerate kinase ( pgk ), triosephosphate isomerase ( tpi ), and enolase ( eno ) genes from Thermotoga neapolitana have been cloned and expressed in Escherichia coli . In high copy number, the pgk gene complemented an E. coli pgk ⁻ strain. In T. neapolitana , the pgk and tpi genes appear to be fused and eno is near those genes. Like T. maritima , T. neapolitana produces phosphoglycerate kinase as both an individual enzyme and a fusion protein with triosephosphate isomerase, and triosephosphate isomerase activity is not found without associated phosphoglycerate kinase activity. Unlike T. maritima , which forms only a 70-kDa fusion protein, T. neapolitana expresses both 73-kDa and 81-kDa isozymes of this fusion protein. These isozymes are present in both T. neapolitana cells and in E. coli cells expressing T. neapolitana genes.     

原核生物eno基因在系统进化中应用及水平转移分析

多基因系统发育研究方法是系统发育分析中的一个重要手段,基因树冲突已成为分子系统发育研究中日益突出的问题。烯醇化酶基因(eno)及其编码的蛋白广泛存在于五界系统中,烯醇化酶为糖酵解途径中重要酶类。文章选取原核生物已注释的eno基因序列进行了系统发育分析。对其中的138个模式菌株的eno基因序列进行系统发育分析和同源性搜索,发现19个模式菌株的eno基因是通过水平转移而来;并通过核苷酸组成、密码子偏好性和基因排列等基因特征分析,进一步验证了水平转移基因的外源性。结果表明:原核生物eno序列具有较高保守性,其大小适中,是研究原核生物系统发育的良好材料。文章在对基因水平转移的供体和受体菌株生活习性、进化历史以及烯醇化酶的结构和功能的研究过程中提供重要参考价值。     

一种新的DyP-type过氧化物酶在大肠杆菌中的重组表达、纯化及鉴定

染料脱色过氧化物酶(DyP-type过氧化物酶)是含有亚铁血红素,能降解各种有毒染料的一类蛋白.为了研究运动发酵单胞菌Zymomonas mobilis ZM4 (ATCC 31821)中一种新的DyP-type过氧化物酶的特点和功能,以Z.mobilis基因组DNA为模板,通过PCR扩增目的基因,克隆到大肠杆菌表达载体pET-21b(+)中.通过ZmDyP与其他DyP-type过氧化物酶的比对,发现它们存在着共同保守氨基酸D149、R239、T254、F256和GXXDG结构基序,说明ZmDyP是Dyp-type过氧化物酶家族的一个新成员.经IPTG诱导大肠杆菌中pET21 b(+)-ZmDyP表达,并将表达的酶进行金属螯合层析纯化.SDS-PAGE分析表明,纯酶分子量为36 kDa,而活性染色显示分子量为108 kDa,表明该酶在活性状态下可能是一个三聚体.光谱扫描显示ZmDyP有一个典型的亚铁血红素吸收峰,说明它是含有亚铁血红素的蛋白.对ZmDyP性质进行了研究,发现以2,2-二氨-双(3-乙基苯并噻唑-6-磺酸)ABTS为底物,ZmDyP表现出更高的转化效率.这些研究结果丰富了DyP-type 过氧化物酶家族信息,并且为ZmDyP的结构功能和反应机制研究奠定了基础.     

A relationship between asparagine synthetase A and aspartyl tRNA synthetase.

A highly conserved protein motif characteristic of Class II aminoacyl tRNA synthetases was found to align with a region of Escherichia coli asparagine synthetase A. The alignment was most striking for aspartyl tRNA synthetase, an enzyme with catalytic similarities to asparagine synthetase. To test whether this sequence reflects a conserved function, site-directed mutagenesis was used to replace the codon for Arg298 of asparagine synthetase A, which aligns with an invariant arginine in the Class II aminoacyl tRNA synthetases. The resulting genes were expressed in E. coli, and the gene products were assayed for asparagine synthetase activity in vitro. Every substitution of Arg298, even to a lysine, resulted in a loss of asparagine synthetase activity. Directed random mutagenesis was then used to create a variety of codon changes which resulted in amino acid substitutions within the conserved motif surrounding Arg298. Of the 15 mutant enzymes with amino acid substitutions yielding soluble enzyme, 13 with changes within the conserved region were found to have lost activity. These results are consistent with the possibility that asparagine synthetase A, one of the two unrelated asparagine synthetases in E. coli, evolved from an ancestral aminoacyl tRNA synthetase.