The requirement for flavine adenine dinucleotide in the formation of acetolactate by Salmonellatyphimurium extracts

Escherichia coli K-12 has two acetohydroxy acid synthase (AHAS) isozymes (AHAS I and AHAS III). Both of these isozymes catalyze the synthesis of a-aceto-a-hydroxybutyrate and a-acetolactate, which are key intermediates of the isoleucine-valine biosynthetic pathway. Strains lacking either isozyme but not both activities have been previously shown to grow well in minimal media in the absence of isoleucine and valine on any of several commonly used carbon sources (e.g., glucose or succinate). We report the characterization of mutants that were unable to grow on either acetate or oleate as a sole carbon source due to a defect in isoleucine-valine biosynthesis. The defect in isoleucine-valine biosynthesis was expressed only on these carbon sources and was due to the loss of AHAS I activity, resulting from lesions in the ilvBN operon. Previously identified ilvBN mutant strains also failed to grow on acetate or oleate minimal media. Our results indicated that AHAS I is an essential enzyme for isoleucing and valine biosynthesis when E. coli K-12 is grown on acetate or oleate as the sole carbon source. AHAS III was expressed during growth on acetate or oleate but was somehow unable to produce sufficient amounts of a-aceto-a-hydroxybutyrate and aL-acetolactate to allow growth.Escherichia coli K-12 possesses the genetic capacity to express three acetohydroxy acid synthase (AHAS) isozymes. AHAS activity is required in E. coli K-12 and in other enteric bacteria for the biosynthesis of a-aceto-ahydroxybutryate for the isoleucine biosynthesis and for the biosynthesis of a-acetolactate for valine biosynthesis ( Fig. 1) (for a review see reference 46). Strains deficient in either AHAS I activity or AHAS III activity are able to synthesize sufficient amounts of isoleucine and valine for growth on minimal media supplemented with various carbon sources (e.g., glucose, glycerol, and succinate) (15, 23). Since strains lacking both AHAS I activity and AHAS III activity require isoleucine and valine for growth (46) and the AHAS activity measured in extracts is valine sensitive (45), it has been assumed that the valine-resistant AHAS isozyme (AHAS II) encoded by ilvG is not expressed in E. coli K-12, unless an ilvO mutation is present (8). However, recent results of Berg et al. (4) suggest that a truncated protein might be produced. AHAS I has recently been purified and is composed of two subunits, which have molecular weights of 60,000 (17, 22) and 11,000 (17). The ilvB gene and a recently identified promoter-distal gene, ilvN, have been shown to encode the AHAS I large and small subunits, respectively (18, 50). AHAS III is also composed of two proteins, which have molecular weights of 60,000 and 18,000 (28) and are encoded by the genes of the ilvIH operon (13,40). A comparison of the polypeptide sequences of AHAS I and AHAS III has shown that both the large subunits and the small subunits of these two isozymes are evolutionarily related to each other (18, 50) and also to the cryptic isozyme, AHAS 11 (18,40,50). Despite the homology b...

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“…AHAS activity was determined by measuring acetolactate formation as previously described (3,43). Assays were done at pH 6.6 and 8.0.…”

Section: Methodsmentioning

confidence: 99%

Acetohydroxy acid synthase I, a required enzyme for isoleucine and valine biosynthesis in Escherichia coli K-12 during growth on acetate as the sole carbon source

Dailey

Cronan

1986

J Bacteriol

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“…E. coli strain CU2001, a rbs-derivative of strain K-12 (prepared by D. McGilvray), was employed as the parent strain. A mineral salts-glucose medium (minimal medium) previously described (12) was used, with supplements as described below, for all studies requiring cell growth.…”

Section: Methodsmentioning

confidence: 99%

Defective Transamination, a Mechanism for Resistance to Ketomycin in Escherichia coli

Jackson

Umbarger

1973

Antimicrob Agents Chemother

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A spontaneous mutant of a derivative of Escherichia coli strain K-12 resistant to 50 ug of ketomycin per ml was selected. The mutant displayed a two-to threefold derepression of the isoleucine-valine biosynthetic enzymes and a reduced growth rate in minimal medium. The lesion was found to lie in the gene (ilvE) specifying transaminase B and resulted in an isoleucine limitation. The presence of exogenous isoleucine during growth in minimal medium restored normal phenotypic properties. The reduced transaminase B activity is responsible for the resistance to ketomycin. An unusual derepression of the acetohydroxy acid synthetase in response to an isoleucine limitation was noted.

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“…The following enzymes were assayed as referenced: (20) 3-isopropylmalate dehydrogenase (IPMP; 2-hydroxy-4-methyl-3-carboxyvaleratenicotinamide adenine dinucleotide oxidoreductase. E. C. 1.1.1.85) (21).…”

Section: Enzymes Assaysmentioning

confidence: 99%

Leucine binding protein and regulation of transport in E. coli

et al. 1977

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Leucine is transported into E. coli cells by high‐affinity transport systems (LIV‐I and leucine‐specific systems) which are sensitive to osmotic shock and require periplasmic binding proteins. In addition leucine is transported by a low‐affinity system (LIV‐II) which is membrane bound and retained in membrane vesicle preparations. The LIV‐I system serves for threonine and alanine in addition to the 3 branched‐chain amino acids. The LIV‐II system is more specific for leucine, isoleucine, and valine while the high‐affinity leucine‐specific system has the greatest specificity. A regulatory locus, livR at minute 22 on the E. coli chromosome produces negatively regulated leucine transport and synthesis of the binding proteins. Valine‐resistant strains have been selected to screen for transport mutants. High‐affinity leucine transport mutants that have been identified include a LIV‐binding protein mutant, livJ, a leucine‐specific binding protein mutant livK and a nonbinding protein component of the LIV‐I system, livH. A fourth mutant, livP, appears to be required only for the low‐affinity LIV‐II system. The existence of this latter mutant indicates that LIV‐I and LIV‐II are parallel transport systems. The 4 mutations concerned with high‐affinity leucine transport form a closely linked cluster of genes on the E. coli chromosome at minute 74. The results of recent studies on the regulation of the high‐affinity transport systems suggests that an attenuator site may be operative in its regulation. This complex regulation appears to require a modified leucyl‐tRNA along with the transcription termination factor rho. Regulation of leucine transport is also defective in relaxed strains. Among the branched‐chain amino acids only leucine produces regulatory changes in LIV‐I activity suggesting a special role of this amino acid in the physiology of E. coli. It was shown that the rapid exchange of external leucine for intracellular isoleucine via the LIV‐I system could create an isolucine pseudoauxotrophy and account for the leucine sensitivity of E. coli.

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The requirement for flavine adenine dinucleotide in the formation of acetolactate by Salmonellatyphimurium extracts

Cited by 110 publications

References 6 publications

Acetohydroxy acid synthase I, a required enzyme for isoleucine and valine biosynthesis in Escherichia coli K-12 during growth on acetate as the sole carbon source

Acetohydroxy acid synthase I, a required enzyme for isoleucine and valine biosynthesis in Escherichia coli K-12 during growth on acetate as the sole carbon source

Defective Transamination, a Mechanism for Resistance to Ketomycin in Escherichia coli

Leucine binding protein and regulation of transport in E. coli

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