Alternative pathways for biosynthesis of leucine and other amino acids in Bacteroides ruminicola and Bacteroides fragilis.
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Bacteroides ruminicola is one of several species of anaerobes that are able to reductively carboxylate isovalerate (or isovaleryl-coenzyme A) to synthesize alpha-ketoisocaproate and thus leucine. When isovalerate was not supplied to growing B. ruminicola cultures, carbon from [U-14C]glucose was used for the synthesis of leucine and other cellular amino acids. When unlabeled isovalerate was available, however, utilization of [U-14C]glucose or [2-14C]acetate for leucine synthesis was markedly and specifically reduced. Enzyme assays indicated that the key enzyme of the common isopropylmalate (IPM) pathway for leucine biosynthesis, IPM synthase, was present in B. ruminicola cell extracts. The specific activity of IPM synthase was reduced when leucine was added to the growth medium but was increased by the addition of isoleucine plus valine, whereas the addition of isovalerate had little or no effect. The activity of B. ruminicola IPM synthase was strongly inhibited by leucine, the end product of the pathway. It seems unlikely that the moderate inhibition of the enzyme by isovalerate adequately explains the regulation of carbon flow by isovalerate in growing cultures. Bacteroides fragilis apparently also uses either the isovalerate carboxylation or the IPM pathway for leucine biosynthesis. Furthermore, both of these organisms synthesize isoleucine and phenylalanine, using carbon from 2-methylbutyrate and phenylacetate, respectively, in preference to synthesis of these amino acids de novo from glucose. Thus, it appears that these organisms have the ability to regulate alternative pathways for the biosynthesis of certain amino acids and that pathways involving reductive carboxylations are likely to be favored in their natural habitats. (+info)
Construction and characterization of Salmonella typhimurium strains that accumulate and excrete alpha- and beta-isopropylmalate.
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Two Salmonella typhimurium strains, which could be used as sources for the leucine biosynthetic intermediates alpha- and beta-isopropylmalate were constructed by a series of P22-mediated transductions. One strain, JK527 [flr-19 leuA2010 Delta(leuD-ara)798 fol-162], accumulated and excreted alpha-isopropylmalate, whereas the second strain, JK553 (flr-19 leuA2010 leuB698), accumulated and excreted alpha- and beta-isopropylmalate. The yield of alpha-isopropylmalate isolated from the culture medium of JK527 was more than five times the amount obtained from a comparable volume of medium in which Neurospora crassa strain FLR(92)-1-216 (normally used as the source for alpha- and beta-isopropylmalate) was grown. Not only was the yield greater, but S. typhimurium strains are much easier to handle and grow to saturation much faster than N. crassa strains. The combination of the two regulatory mutations flr-19, which results in constitutive expression of the leucine operon, and leuA2010, which renders the first leucine-specific biosynthetic enzyme insensitive to feedback inhibition by leucine, generated limitations in the production of valine and pantothenic acid. The efficient, irreversible, and unregulated conversion of alpha-ketoisovaleric acid into alpha-isopropylmalate (alpha-isopropylmalate synthetase K(m) for alpha-ketoisovaleric acid, 6 x 10(-5) M) severely restricted the amount of alpha-ketoisovaleric acid available for conversion into valine and pantothenic acid (ketopantoate hydroxymethyltransferase K(m) for alpha-ketoisovaleric acid, 1.1 x 10(-3) M; transaminase B K(m) for alpha-ketoisovaleric acid, 2 x 10(-3) M). (+info)
Inactivation of yeast alpha-isopropylmalate synthase by CoA. Antagonism between CoA and adenylates and the mechanism of CoA inactivation.
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Yeast alpha-isopropylmalate synthase (EC 4.1.3.12) is inactivated by micromolar concentrations of CoA in the presence of Zn2+. We report here that rapid reactivation of inactivated enzyme (full recovery in less than 10 min) occurred in the presence of millimolar concentrations of ATP or ADP, using permeabilized cells. With purified, CoA-inactivated enzyme, ATP had only a weak reactivating effect which increased drastically, however, when a chelator was added at a concentration (0.1 mM) which by itself had little effect. Higher concentrations of chelator (1 mM) caused rapid reactivation even in the absence of ATP. Reactivation was also possible by removing CoA from equilibrium with oxidized glutathione, with acetyl phosphate in the presence of phosphotransacetylase, or by dialysis; however, these processes were very slow. Protection against CoA inactivation of alpha-isopropylmalate synthase was provided by high concentrations of ATP and, to a much lesser extent, ADP, by a high adenylate energy charge, by chelators, and by 3'-dephospho-CoA. Enzyme which had been inactivated with [3H]CoA did not retain any radioactivity (above control) when extracted with phenol. This result, together with other observations, is interpreted to mean that inactivation does not involve covalent modification, but is more likely the result of the formation of an enzyme.CoA.zinc complex held together by noncovalent forces. The physiological significance of the CoA effect is discussed. (+info)
Evidence that alpha-isopropylmalate synthase of Saccharomyces cerevisiae is under the "general" control of amino acid biosynthesis.
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The specific activity and the immunoreactive amount of alpha-isopropylmalate synthase were more than three times above wild-type values in a Saccharomyces cerevisiae mutant (cdr1) with constitutively derepressed levels of enzymes known to be under the "general" control of amino acid biosynthesis. The specific activity was also higher in lysine- and arginine-leaky strains when these were grown under limiting conditions, and in wild-type cells grown in the presence of 5-methyltryptophan. A low specific activity was found in a mutant (ndr1) unable to derepress enzymes of the general control system. Neither isopropylmalate isomerase nor beta-isopropylmalate dehydrogenase responded to general control signals. (+info)
Additive activation of yeast LEU4 transcription by multiple cis elements.
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The LEU4 gene of Saccharomyces cerevisiae and the enzyme encoded by LEU4, alpha-isopropylmalate synthase, occupy a special position in amino acid metabolism. alpha-Isopropylmalate synthase catalyzes the first committed step in leucine biosynthesis. However, the reaction product alpha-isopropylmalate is not only an intermediate in the leucine biosynthetic pathway, but also functions as co-activator of at least six genes, both within and outside of the leucine pathway. The metabolic importance of alpha-isopropylmalate appears to be reflected in the surprisingly multifaceted regulation of LEU4 expression. This report describes an analysis of functional cis elements in the LEU4 promoter. Five such elements were identified. Three distal elements, designated UASLEU, GCE-A, and GCE-B, are responsible for regulation by the regulatory proteins Leu3p and Gen4p, respectively. The incremental activation of LEU4 by these elements is additive and independent. In addition, two proximal elements were localized. One of these conforms to the TATA consensus sequence and exhibits high affinity for TATA binding protein. The other element shows strong sequence identity with the Bas2p binding site and appears to be involved in basal and phosphate-mediated regulation of LEU4. (+info)
Biosynthesis of norvaline, norleucine, and homoisoleucine in Serratia marcescens.
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The biosynthetic pathways of norvaline homoisoleucine were examined using regulatory mutants of leucine biosynthesis in Serratia marcescens. alpha-Isopropylmalate synthetase [EC 4.1.3.12], the first enzyme of leucine biosynthesis, catalyzed the condensations of acetyl-CoA with pyruvate, alpha-ketobutyrate, alpha-ketovalerate, or alpha-keto-beta-methylvalerate as well as alpha-ketoisovalerate. These condensations were inhibited by leucine in the alpha-aminobutyrate-resistant mutant, a mutant with derepressed leucine biosynthetic enzymes. However, these condensations were coordinately desensitized in the isoleucine leaky revertant, a leucine accumulator. The formation of norvaline or homoisoleucine was greater in the leucine accumulator, but its leucine auxotroph did not form these unnatural amino acids. Thus, norvaline and homoisoleucine are considered to be formed from alpha-ketobutyrate and alpha-keto-beta-methylvalerate by the leucine biosynthetic enzymes. This view was confirmed by the findings that a norvaline accumulator could be obtained by derivation of the leucine accumulator into an isoleucine-valine auxotroph. Norleucine was also found to be formed from alpha-ketovalerate, an alpha-ketoacid corresponding to norvaline. (+info)
Regulation of tryptophan biosynthesis in Methanobacterium thermoautotrophicum Marburg.
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A tryptophan-auxotrophic mutant of the archaeon Methanobacterium thermoautotrophicum Marburg was grown with growth-promoting and growth-limiting concentrations of tryptophan. The specific activities of anthranilate synthase (TrpEG) and tryptophan synthase (TrpB) increased 30- to 40-fold in tryptophan-starved cells. Levels of trpE-specific and trpD-specific mRNAs (transcripts of the first and the last genes, respectively, of the M. thermoautotrophicum Marburg trp gene cluster) increased about 10-fold upon starvation for tryptophan. Thus, the expression of the trp genes appears to be regulated primarily at the level of transcription. These data support transcription of trp genes as an operon and support a regulatory model involving a repressor. Anthranilate synthase was feedback inhibited by L-tryptophan, with a Ki of 3.0 microM. In a leucine-auxotrophic mutant starved for L-leucine, the level of alpha-isopropylmalate synthase (LeuA) was 10-fold higher than in cells grown with L-leucine. In addition to the finding of specific regulation of gene expression by the end products of their respective pathways, it was found that the levels of anthranilate synthase and alpha-isopropylmalate synthase were reduced upon growth in the presence of amino acids of other families, such as L-alanine, L-proline, or L-arginine. Conversely, starvation for tryptophan caused a slight elevation of alpha-isopropylmalate synthase and starvation for leucine caused a significant increase of anthranilate synthase and tryptophan synthase specific activities. The latter effect was also observed at the level of trp-specific mRNA and is reminiscent of general amino acid control. (+info)
Properties of some norvaline-resistant mutants of Bacillus subtilis.
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DL-Norvaline inhibits growth of wild-type Bacillus subtilis. A number of mutants resistant to growth inhibition by this analogue were isolated and studied. Cross-feeding experiments and paper chromatography of culture supernatants indicated that the mutants excreted leucine and possibly valine and glutamate. Enzymic analysis indicated that the mutants were derepressed for acetohydroxy-acid synthetase and alpha-isopropylmalate synthetase; however, no derepression of threonine deaminase, dihydroxyacid dehydrase or transaminase B was observed. (+info)