Some catalytic and molecular properties of threonine deaminase from Bacillus stearothermophilus.
(1/110)
Threonine deaminase [EC 4.2.1.16] was highly purified from Bacillus stearothermophilus. The enzyme exhibited maximum activity at 65 degrees and at pH 9.2--9.6. It was inactivated on dilution and on storage at 4 degrees, but was protected by egg albumin. The enzyme was labile at 65 degrees, but became stable in the presence of egg albumin and isoleucine at pH 7.0. The substrate saturation curve for the enzyme reaction at 40 or 65 degrees was hyperbolic, but in the presence of isoleucine, the curve became sigmoidal (n = 2). The enzyme was more sensitive to isoleucine at 40 degrees than at 65 degrees, while valine slightly inhibited the enzyme at both 40 and 65 degrees. Inhibition of the enzyme by isoleucine was antagonized by valine at 40 and 65 degrees. These properties were essentially similar to those of the enzymes from mesophilic and thermophilic bacteria. The enzyme existed in two forms with different molecular sizes, 1.5-5 X 10(6) and 2 X 10(5) daltons, at pH 7.0 and at temperatures below 40 degrees. The larger component disaggregated into the small one at pH 8.5 or above, at temperatures above 50 degrees or in the presence of isoleucine and valine. (+info)
Construction of an L-isoleucine overproducing strain of Escherichia coli K-12.
(2/110)
The genes for a threonine deaminase that is resistant to feedback inhibition by L-isoleucine and for an active acetohydroxyacid synthase II were introduced by a plasmid into a L-threonine-producing recombinant strain of Escherichia coli K-12. Analysis of culture broth of the strain using 13C nuclear magnetic resonance suggested that alpha, beta-dihydroxy-beta-methylvalerate (DHMV) and alpha-keto-beta-methylvalerate (KMV), the third and the fourth intermediates in the L-isoleucine biosynthetic pathway from L-threonine, respectively, accumulated in the medium in amounts comparable to that of L-isoleucine. The ratio of accumulated L-isoleucine:DHMV:KMV were approximately 2:1:1. The concentration of accumulated L-isoleucine increased by twofold after the additional introduction of the genes for dihyroxyacid dehydratase (DH) and transaminase-B (TA-B), and the intermediates no longer accumulated. The resultant strain TVD5 accumulated 10 g/l of L-isoleucine from 40 g/l of glucose. (+info)
Expression of the Escherichia coli catabolic threonine dehydratase in Corynebacterium glutamicum and its effect on isoleucine production.
(3/110)
The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1. 16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to alpha-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the tdcB gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by the ilvA gene), fermentations were performed with the wild-type strain, an ilvA-overexpressing strain, and a tdcB-expressing strain. By expressing the heterologous catabolic threonine dehydratase in C. glutamicum, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway. (+info)
The effect of epsilon-aminocaproic acid on biochemical changes in the development of the cellular slime mould Dictyostelium discoideum.
(4/110)
epsilon-Aminocaproic acid (EACA) inhibited the development of Dictyostelium discoideum strain AX2 after the aggregation stage. Biochemical changes that occurred early in development (loss of cellular protein, RNA and carbohydrate; increase in the specific activity of N-acetylglucosaminidase, alpha-mannosidase, threonine deaminase and leucine aminopeptidase) were not affected by concentrations of EACA which blocked development; but biochemical changes that occurred later (synthesis of carbohydrate, increase in the specific activity of UDP-glucose pyrophosphorylase) were inhibited. Spores from fruiting bodies formed in the presence of low concentrations of EACA were larger, more spherical and less able to survive heat treatment than spores from fruiting bodies of control (no EACA) cells. (+info)
Derivation of glycine from threonine in Escherichia coli K-12 mutants.
(5/110)
Escherichia coli AT2046 has been shown previously to lack the enzyme serine transhydroxymethylase and to require exogenous glycine for growth as a consequence. Strains JEV73 and JEV73R, mutants derived from strain AT2046, are shown here to be serine transhydroxymethylase deficient, but able to derive their glycine from endogenously synthesized threonine. Leucine is shown to be closely involved in the regulation of biosynthesis of glycine, to spare glycine in strain AT2046T, to replace glycine in strain JEV73, and to increase threonine conversion to glycine in a representative prototroph of E. coli. An interpretation of strains JEV73 and JEV73R as regulatory mutants of strain AT2046 is given. A hypothesis as to the role of leucine as a signal for nitrogen scavenging is suggested. (+info)
Inhibition of Escherichia coli isoleucine biosynthesis by isoleucine tetrazole.
(6/110)
Growth of a derivative of Escherichia coli K-10 was strongly inhibited by 2 times 10(-4) M L-5(1-amino-2-methylbutyl)-tetrazole (isoleucine tetrazole). Growth inhibition was reversed by isoleucine, threonine, glycyl-L-isoleucine, or glycyl-L-threonine, and, in a valine-resistant mutant, by L-valine. Partial reversal of growth inhibiton was effected by L-leucine, L-methionine, or L-homoserine. The tetrazole inhibited the activity of the biosynthetic threonine deaminase (EC 4.2.1.16 L-threonine hydrolyase [deaminating]), the inhibition being relieved by L-valine. The tetrazole also inhibited isoleucyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.5 L-isoleucine: tRNA ligase [adenosine monophosphate]), but was without effect on the activities of alpha-isopropylmalate synthetase or acetohydroxy acid synthetase. One class of isoleucine tetrazole-resistant mutants produced biosynthetic threonine deaminases which were no longer subject to feedback inhibition by either isoleucine or the tetrazole. (+info)
New approach to analysis of deviations from hyperbolic law in enzyme kinetics.
(7/110)
An empirical equation that describes deviations from Michaelian kinetics is proposed. The equation allows the limiting values of the Michaelis constant at v/Vmax --> 0 and v/Vmax --> 1 to be estimated (v is the rate of the enzymatic reaction and Vmax is the limiting value of v at saturating concentrations of substrate). The applicability of the equation is demonstrated for kinetic data obtained for glutamate dehydrogenases from various sources (negative kinetic cooperativity for coenzyme) and for biosynthetic threonine deaminase from pea seedlings (sharper approaching the limiting value of the enzymatic reaction rate with increasing substrate concentration in comparison with the hyperbolic law). The negative cooperativity for the function of saturation of protein by ligand is also analyzed (data on binding of spin-labeled NAD, NADH, and NADPH by beef liver glutamate dehydrogenase and binding of cupric ions by BSA are used as examples). (+info)
Role of leucyl-tRNA synthetase in regulation of branched-chain amino-acid transport.
(8/110)
The regulation of the transport of leucine, isoleucine, and valine in Escherichia coli B/r was studied in a mutant with a complete deletion of the leucine biosynthetic operon and a temperature-sensitive leucyl-tRNA synthetase [L-leucine:tRNALeu ligase (AMP-forming), EC 6.1.1.4]. Under conditions of excess leucine and a functional leucyl-tRNA synthetase transport activity was repressed. Shifting the culture to a temperature at which the activation of leucine to an appropriate tRNA species became growth-rate-limiting led to a large increase in the high-affinity transport of leucine, isoleucine, and valine (system LIV-I) while the uptake of histidine and proline was unchanged. A similar increase was observed for branched-chain amino-acid binding protein activity. The temperature change did not alter the transport activity for any of these substrates or the level of the binding proteins in an isogenic strain with a normal leucyl-tRNA synthetase. The increase in transport activity observed in the mutant was prevented by inhibitors of protein and RNA synthesis and probably represents an increase in the differential rate of synthesis of the protein(s) required for transport. These experiments demonstrate that the repression of branched-chain amino-acid transport involves the interaction of leucine with its aminoacyl-tRNA synthetase and its cognate leucyl-tRNA species. (+info)