Abstract
A previously recognized open reading frame (T. Yura, H. Mori, H. Nagai, T. Nagata, A. Ishihama, N. Fujita, K. Isono, K. Mizobuchi, and A. Nakata, Nucleic Acids Res. 20:3305-3308) from the 0.2-min region of the Escherichia coli K-12 chromosome is shown to encode a functional transaldolase activity. After cloning of the gene onto high-copy-number vectors, transaldolase B (D-sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate dihydroxyacetone transferase; EC 2.2.1.2) was overexpressed up to 12.7 U mg of protein-1 compared with less than 0.1 U mg of protein-1 in wild-type homogenates. The enzyme was purified from recombinant E. coli K-12 cells by successive ammonium sulfate precipitations (45 to 80% and subsequently 55 to 70%) and two anion-exchange chromatography steps (Q-Sepharose FF, Fractogel EMD-DEAE tentacle column; yield, 130 mg of protein from 12 g of cell wet weight) and afforded an apparently homogeneous protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a subunit size of 35,000 +/- 1,000 Da. As the enzyme had a molecular mass of 70,000 Da by gel filtration, transaldolase B is likely to form a homodimer. N-terminal amino acid sequencing of the protein verified its identity with the product of the cloned gene talB. The specific activity of the purified enzyme determined at 30 degrees C with the substrates fructose-6-phosphate (donor of C3 compound) and erythrose-4-phosphate (acceptor) at an optimal pH (50 mM glycylglycine [pH 8.5]) was 60 U mg-1.Km values for the substrates fructose-6-phosphate and erythrose-4-phosphate were determined at 1,200 and 90 microM, respectively. Kinetic constants for the other two physiological reactants, D,L-glyceraldehyde 3-phosphate (Km, 38 microM; relative activity [V(rel)], 8%) and sedoheptulose-7-phosphate (K(m), 285 microM; V(rel), 5%) were also determined. Fructose acted as a C(3) donor at a high apparent K(m) (>/=M) and with a V(rel) of 12%. The enzyme was inhibited by Tris-HCl, phosphate, or sugars with the L configuration at C(2) (L-glyceraldehyde, D-arabinose-5-phosphate).
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- Banki K., Halladay D., Perl A. Cloning and expression of the human gene for transaldolase. A novel highly repetitive element constitutes an integral part of the coding sequence. J Biol Chem. 1994 Jan 28;269(4):2847–2851. [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Brickman E., Soll L., Beckwith J. Genetic characterization of mutations which affect catabolite-sensitive operons in Escherichia coli, including deletions of the gene for adenyl cyclase. J Bacteriol. 1973 Nov;116(2):582–587. doi: 10.1128/jb.116.2.582-587.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- HORECKER B. L., SMYRNIOTIS P. Z. Purification and properties of yeast transaldolase. J Biol Chem. 1955 Feb;212(2):811–825. [PubMed] [Google Scholar]
- Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
- Iida A., Teshiba S., Mizobuchi K. Identification and characterization of the tktB gene encoding a second transketolase in Escherichia coli K-12. J Bacteriol. 1993 Sep;175(17):5375–5383. doi: 10.1128/jb.175.17.5375-5383.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jacoby J., Hollenberg C. P., Heinisch J. J. Transaldolase mutants in the yeast Kluyveromyces lactis provide evidence that glucose can be metabolized through the pentose phosphate pathway. Mol Microbiol. 1993 Nov;10(4):867–876. doi: 10.1111/j.1365-2958.1993.tb00957.x. [DOI] [PubMed] [Google Scholar]
- Josephson B. L., Fraenkel D. G. Transketolase mutants of Escherichia coli. J Bacteriol. 1969 Dec;100(3):1289–1295. doi: 10.1128/jb.100.3.1289-1295.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kohara Y., Akiyama K., Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. doi: 10.1016/0092-8674(87)90503-4. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lim R., Cohen S. S. D-phosphoarabinoisomerase and D-ribulokinase in Escherichia coli. J Biol Chem. 1966 Oct 10;241(19):4304–4315. [PubMed] [Google Scholar]
- Miosga T., Schaaff-Gerstenschlager I., Franken E., Zimmermann F. K. Lysine144 is essential for the catalytic activity of Saccharomyces cerevisiae transaldolase. Yeast. 1993 Nov;9(11):1241–1249. doi: 10.1002/yea.320091111. [DOI] [PubMed] [Google Scholar]
- Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
- Newman T., de Bruijn F. J., Green P., Keegstra K., Kende H., McIntosh L., Ohlrogge J., Raikhel N., Somerville S., Thomashow M. Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones. Plant Physiol. 1994 Dec;106(4):1241–1255. doi: 10.1104/pp.106.4.1241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- RACKER E., SCHROEDER E. Formation and utilization of octulose-8-phosphate by transaldolase and transketolase. Arch Biochem Biophys. 1957 Jan;66(1):241–243. doi: 10.1016/0003-9861(57)90555-6. [DOI] [PubMed] [Google Scholar]
- Rost B., Sander C. Combining evolutionary information and neural networks to predict protein secondary structure. Proteins. 1994 May;19(1):55–72. doi: 10.1002/prot.340190108. [DOI] [PubMed] [Google Scholar]
- Rost B., Sander C. Prediction of protein secondary structure at better than 70% accuracy. J Mol Biol. 1993 Jul 20;232(2):584–599. doi: 10.1006/jmbi.1993.1413. [DOI] [PubMed] [Google Scholar]
- Schaaff I., Hohmann S., Zimmermann F. K. Molecular analysis of the structural gene for yeast transaldolase. Eur J Biochem. 1990 Mar 30;188(3):597–603. doi: 10.1111/j.1432-1033.1990.tb15440.x. [DOI] [PubMed] [Google Scholar]
- Sprenger G. A. Nucleotide sequence of the Escherichia coli K-12 transketolase (tkt) gene. Biochim Biophys Acta. 1993 Nov 16;1216(2):307–310. doi: 10.1016/0167-4781(93)90161-6. [DOI] [PubMed] [Google Scholar]
- Sprenger G. A., Schörken U., Sprenger G., Sahm H. Transketolase A of Escherichia coli K12. Purification and properties of the enzyme from recombinant strains. Eur J Biochem. 1995 Jun 1;230(2):525–532. doi: 10.1111/j.1432-1033.1995.0525h.x. [DOI] [PubMed] [Google Scholar]
- Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
- Witke C., Götz F. Cloning, sequencing, and characterization of the gene encoding the class I fructose-1,6-bisphosphate aldolase of Staphylococcus carnosus. J Bacteriol. 1993 Nov;175(22):7495–7499. doi: 10.1128/jb.175.22.7495-7499.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yura T., Mori H., Nagai H., Nagata T., Ishihama A., Fujita N., Isono K., Mizobuchi K., Nakata A. Systematic sequencing of the Escherichia coli genome: analysis of the 0-2.4 min region. Nucleic Acids Res. 1992 Jul 11;20(13):3305–3308. doi: 10.1093/nar/20.13.3305. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang M., Eddy C., Deanda K., Finkelstein M., Picataggio S. Metabolic Engineering of a Pentose Metabolism Pathway in Ethanologenic Zymomonas mobilis. Science. 1995 Jan 13;267(5195):240–243. doi: 10.1126/science.267.5195.240. [DOI] [PubMed] [Google Scholar]
- Zhao G., Winkler M. E. An Escherichia coli K-12 tktA tktB mutant deficient in transketolase activity requires pyridoxine (vitamin B6) as well as the aromatic amino acids and vitamins for growth. J Bacteriol. 1994 Oct;176(19):6134–6138. doi: 10.1128/jb.176.19.6134-6138.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]