Purification of pyruvate formate-lyase from Streptococcus mutans and its regulatory properties.

Pyruvate formate-lyase (EC 2.3.1.54) from Streptococcus mutans strain JC2 was purified in an anaerobic glove box, giving a single band on disk and sodium dodecyl sulfate electrophoresis. This enzyme was immediately inactivated by exposure to the air. Enzyme activity was unstable even when stored anaerobically, but the activity was restored by preincubating the inactivated crude enzyme with S-adenosyl-L-methionine, oxamate, and reduced for ferredoxin or methylviologen. On the other hand, the purified enzyme was not reactivated. Either D-glyceraldehyde 3-phosphate or dihydroxyacetone phosphate strongly inhibited this enzyme. The inhibitory effects of these compounds were largely influenced by enzyme concentration. The inhibition of these triose phosphates in cooperation with the reactivating effect of ferredoxin and the fluctuations of both the enzyme and the triose phosphate levels may efficiently regulate the pyruvate formate-lyase activity in S. mutans in vivo.     

Carbon metabolism regulates expression of the pfl (pyruvate formate-lyase) gene in Escherichia coli.

The anaerobic expression of pfl is reduced both in a strain mutated in the pgi gene and in a pfkA pfkB double mutant strain when cells are grown in medium supplemented with glucose. When cells are grown in medium supplemented with either fructose or pyruvate, no reduction is observed in these strains. The amount of pyruvate in the cells may be responsible for the reduced expression of pfl in the strains mutated in the genes encoding the glycolytic enzymes. Because of the lowered oxygen concentration in the medium, the expression of pfl is induced when an exponentially growing culture enters the stationary phase. This induction is increased when the Casamino Acid concentration is raised 10-fold or when the medium is supplemented with NaCl. Superhelicity of DNA is decreased in a pgi mutant strain grown in medium supplemented with glucose. The superhelicity is also changed, but the opposite way, in a wild-type strain grown in medium supplemented with Casamino Acids at a high concentration or 0.3 M sodium chloride. Our data show that changes in superhelicity do not affect the aerobic expression of pfl but might be important for the anaerobic induction of pfl.     

Expression of pyruvate formate-lyase of Escherichia coli from the cloned structural gene

It is shown here that a plasmid (p29) derived from the transducing phage aspC2 (Christiansen and Pedersen 1981) codes for pyruvate formate-lyase. The identity of the 80 kilodaltons (kd) gene product of plasmid p29 with the pyruvate formate-lyase polypeptide was proven (i) by comigration of the gene product expressed in the maxicell system with purified enzyme on O'Farrell gels, and (ii) by comparison of the peptide maps obtained from limited proteolysis. In vivo the 80 kd form of the enzyme was proteolytically converted to a 78 kd polypeptide. The two polypeptides (80 kd and 78 kd) and their charge isomers present in purified enzyme preparations are therefore products of a single gene.Aerobically grown cells of Escherichia coli contained a basal level of pyruvate formate-lyase which was derepressed 5-to 10-fold under anaerobiosis. Derepression also occurred during anaerobic growth on glycerol plus fumarate. Presence of plasmid p29 caused overproduction of pyruvate formatelyase, 11-fold upon anaerobic growth on glucose, 14-fold upon aerobic growth on glucose and 33-fold upon aerobic growth at the expense of D-lactate.Non-Standard Abbreviation MOPS 4-morpholine-propane sulfonic     

大肠杆菌yigP基因转录调控序列的鉴定

【目的】调查yigP基因启动子的活性,并对该转录调控序列进行分析。【方法】以lacZ为报告基因,克隆启动子片段至启动子探针质粒中,通过检测β-半乳糖苷酶活性判断启动子活性,并通过克隆一系列逐步缩短的启动子片段来确定启动子所在区域。利用定点突变技术,对启动子的重要序列进行定点突变,调查其对启动子活性的影响。【结果】确定了yigP基因启动子的区域,鉴定了启动子的-10区和-35区,并发现了启动子上游存在一个负调控序列,对该序列进行了初步的研究显示其中部分序列是这种负调控作用的核心序列。【结论】对yigP基因的转录调控序列进行了鉴定,丰富了我们对基因转录调控的认识。     

A negative transcriptional control element located upstream of the murine c-myc gene

[This corrects the article on p. 899 in vol. 5, PMID: 3013622.].     

Primary structures of Escherichia coli pyruvate formate-lyase and pyruvate-formate-lyase-activating enzyme deduced from the DNA nucleotide sequences

The structural gene of pyruvate formate-lyase (pfl) and that of pyruvate-formate-lyase-activating enzyme were shown to be adjacent on the chromosomal map of Escherichia coli. DNA sequencing was performed along a stretch of 3592 nucleotides to obtain the amino acid sequences of both proteins. The derived primary structures (759 and 245 residues) were confirmed by partial structure analyses on the purified proteins. The open reading frames are separated by a 194-nucleotide stretch, and their flanking regions include signal elements that are compatible with separate control of protein synthesis from the two genes.