TRANSALDOLASE-B OF ESCHERICHIA-COLI K-12 - CLONING OF ITS GENE, TALB,AND CHARACTERIZATION OF THE ENZYME FROM RECOMBINANT STRAINS

A previously recognized open reading frame (T. Yura, H. Mori, H. Nagai , T. Nagata, A. Ishihama, N. Fujita, K. Isono, K, Mizobuchi, and A, Na kata, 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 trans aldolase activity. After cloning of the gene onto high-copy-number vec tors, transaldolase B (D-sedoheptulose-7-phosphate:D glycerald phate d ihydroxyacetone 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, co li K-12 cells by successive ammonium sulfate precipitations (45 to 80% and subsequently 55 to 70%) and two anion-exchange chromatography ste ps (Q-Sepharose FF, Fractogel EMD-DEAE tentacle column; yield, 130 mg of protein from 12 g of cell wet weight) and afforded an apparently ho mogeneous protein band on sodium dodecyl sulfate-polyacrylamide gel el ectrophoresis,vith a subunit size of 35,000 +/- 1,000 Da. As the enzym e 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 th e protein verified its identity with the product of the cloned gene ta lB. The specific activity of the purified enzyme determined at 30 degr ees C with the substrates fructose-6-phosphate (donor of C-3 compound) and erythrose 4 phosphate (acceptor) at an optimal pH (50 mM glycylgl ycine [pH 8.5]) was 60 U mg(-1). K-m values for the substrates fructos e-6-phosphate and erythrose 4-phosphate were determined at 1,200 and 9 0 mu M, respectively, Kinetic constants for the other two physiologica l reactants, D,L-glyceraldehyde 3 phosphate (K-m, 38 mu M; relative ac tivity [V-rel], 8%) and sedoheptulose-7-phosphate (K-m, 285 mu M; V-re l 5%) were also determined, Fructose acted as a C-3 donor at a high ap parent K-m (greater than or equal to 22 M) and with a V-rel of 12%. Th e enzyme was inhibited by Tris-HCl, phosphate, or sugars with the L co nfiguration at C-2 (L glyceraldehyde, D-arabinose-5-phosphate).