Identification and characterization of the tktB gene encoding a second transketolase in Escherichia coli K-12

Abstract

We isolated a transposon TnlO insertion mutant of Escherichia coli K-12 which could not grow on MacConkey plates containing D-ribose. Characterization of the mutant revealed that the level of the transketolase activity was reduced to one-third of that of the wild type. The mutation was mapped at 63.5 min on the E. coli genetic map, in which the transketolase gene (tkt) had been mapped. A multicopy suppressor gene which complemented the tkt mutation was cloned on a 7.8-kb PstI fragment. The cloned gene was located at 53 min on the chromosome. Subcloning and sequencing of a 2.7-kb fragment containing the suppressor gene identified an open reading frame encoding a polypeptide of 667 amino acids with a calculated molecular weight of 72,973. Overexpression of the protein and determination of its N-terminal amino acid sequence defined unambiguously the translational start site of the gene. The deduced amino acid sequence showed similarity to sequences of transketolases from Saccharomyces cerevisiae and Rhodobacter sphaeroides. In addition, the level of the transketolase activity increased in strains carrying the gene in multicopy. Therefore, the gene encoding this transketolase was designated tktB and the gene formerly called tkt was renamed tktA. Analysis of the phenotypes of the strains containing tktA, tktB, or tktA tktB mutations indicated that tktA and tktB were responsible for major and minor activities, respectively, of transketolase in E. coli.Transketolase (EC 2.2.1.1) catalyzes transfer of the glycol aldehyde moiety from a ketose or its phosphate to an aldose or its phosphate. The enzyme is found in animals, plants, and bacteria and is involved in the pentose phosphate pathway responsible for production of NADPH and several sugar phosphate intermediates such as ribose 5-phosphate, erythrose 4-phosphate, and sedoheptulose 7-phosphate. Ribose 5-phosphate is utilized for the biosynthesis of purine and pyrimidine nucleotides and histidine, erythrose 4-phosphate is used for the biosynthesis of aromatic amino acids, and sedoheptulose 7-phosphate is used for the biosynthesis of cell wall components in gram-negative bacteria. The reaction is also important in CO2 fixation in photosynthetic organisms.In Escherichia coli Josephson and Fraenkel (18,19) first isolated mutants defective in transketolase activity. These mutants were unable to use L-arabinose or D-xylose as a sole carbon source and required shikimic acid or aromatic amino acids for growth on a minimal medium. The requirement for aromatic amino acids was shown to be slightly leaky, and the existence of low residual transketolase activity was suggested by the authors. The mutations were mapped around 62 min on the E. coli chromosome. A similar mutant was also isolated from Salmonella typhimurium, and the role of transketolase in supplying sedoheptulose 7-phosphate was examined (7). In this report, we describe isolation of a transposon TnlO insertion mutant of E. coli defective in transketolase as a ribose-sensitive mutant on MacConkey plates containing D-ri...

Properties and functions of the thiamin diphosphate dependent enzyme transketolase

“…Genes coding for DHAS from the methanolutilising yeast Hansenula polymorpha [70] [132,155] and E. coli [68,152]; three di erent forms of TK have been cloned from the desiccation-tolerant plant Craterostigma plantagineum [4].…”

“…This was the ®rst convincing evidence for genetic heterogeneity of any TK. Since then two genetic variants have been found in the bacterium E. coli [68,152] and three in the plant C. plantagineum [4]. While it is still unclear why E. coli and S. cerevisiae have two di erent TKs it could be shown that only one of the three TK forms in C. plantagineum is expressed constitutively.…”

Molecular Evolutionary Analysis of the Thiamine-Diphosphate-Dependent Enzyme, Transketolase

“…Since all of these substitutions occur in a single block, we wonder whether compounded sequencing errors may provide a simple explanation for the discrepancy. However, consideration must also be given to the fact that several organisms have more than one transketolase gene (e.g., E. coli (Sprenger 1993;Iida et al 1993), S. cerevisiae (Schaaf-Gerstenschläger and Zimmermann 1993; Sundström et al 1993), and C. plantagineum (Bernacchia et al 1995), and the possibility of two transketolase genes in humans has not been ruled out.…”

“…A second transketolase, distinct from but closely related to the first, has been cloned in both Escherichia coli (Iida et al 1993) and S. cerevisiae (Schaaff-Gerstenschläger and Zimmermann 1993), although any unique functions and metabolic implications of these enzymes remain unclear. In the dessication-tolerant plant Craterostigma plantagineum, three distinct forms of transketolase have been cloned (Bernacchia et al 1995).…”

Chloroplast pentose‐5‐phosphate 3‐epimerase from potato: cloning, cDNA sequence, and tissue‐specific enzyme accumulation

“…This may indicate the presence in E. coli of another protein (YJCU) which is functionally equivalent to epimerase. It is remarkable to note, that other proteins of the oxidative pentose phosphate cycle, transketolase and ribose-5-phosphate isomerase, are also present as two isoforms in E. coli [16,17]. Fig.…”