Bacterial catabolism of threonine. Threonine degradation initiated by L-threonine-NAD+ oxidoreductase

Bell, Steven; Turner, J M

doi:10.1042/bj1560449

Cited by 44 publications

(26 citation statements)

References 32 publications

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“…An L-threonine 3-dehydrogenase activity (EC 1.1.1.103) (tdh in E. coli) is considered responsible for the degradation of L-threonine in Corynebacterium sp. strain B6 (2). However, this enzyme activity is not detectable in C. glutamicum ATCC 13032.…”

Section: Discussionmentioning

confidence: 87%

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Identification of glyA (Encoding Serine Hydroxymethyltransferase) and Its Use Together with the Exporter ThrE To Increase l -Threonine Accumulation by Corynebacterium glutamicum

Šimić

Willuhn

Sahm

et al. 2002

Appl Environ Microbiol

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L-Threonine can be made by the amino acid-producing bacterium Corynebacterium glutamicum. However, in the course of this process, some of the L-threonine is degraded to glycine. We detected an aldole cleavage activity of L-threonine in crude extracts with an activity of 2.2 nmol min ؊1 (mg of protein) ؊1 . In order to discover the molecular reason for this activity, we cloned glyA, encoding serine hydroxymethyltransferase (SHMT). By using affinity-tagged glyA, SHMT was isolated and its substrate specificity was determined. The aldole cleavage activity of purified SHMT with L-threonine as the substrate was 1.3 mol min ؊1 (mg of protein)؊1 , which was 4% of that with L-serine as substrate. Reduction of SHMT activity in vivo was obtained by placing the essential glyA gene in the chromosome under the control of P tac , making glyA expression isopropylthiogalactopyranoside dependent. In this way, the SHMT activity in an L-threonine producer was reduced to 8% of the initial activity, which led to a 41% reduction in glycine, while L-threonine was simultaneously increased by 49%. The intracellular availability of L-threonine to aldole cleavage was also reduced by overexpressing the L-threonine exporter thrE. In C. glutamicum DR-17, which overexpresses thrE, accumulation of 67 mM instead of 49 mM L-threonine was obtained. This shows that the potential for amino acid formation can be considerably improved by reducing its intracellular degradation and increasing its export.

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Section: Discussionmentioning

confidence: 87%

“…(mg of protein) Ϫ1 was detected (2). Furthermore, we wanted to reduce an identified activity in C. glutamicum and to examine whether improved L-threonine accumulation occurs.…”

mentioning

confidence: 99%

Identification of glyA (Encoding Serine Hydroxymethyltransferase) and Its Use Together with the Exporter ThrE To Increase l -Threonine Accumulation by Corynebacterium glutamicum

Šimić

Willuhn

Sahm

et al. 2002

Appl Environ Microbiol

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“…Bacteria were maintained on nutrient broth and cultivated on semisynthetic media as described for similar soil isolates (Bell & Turner, 1976a). Threonine medium contained 2g of L-threonine/litre plus mineral salts supplemented with 0.05 % yeast extract or 2mM-L-valine.…”

Section: Materials and Methods Micro-organisms And Mediamentioning

confidence: 99%

“…The product of this oxidation, 2-amino-3-oxobutyrate, is cleaved to acetyl-CoA and glycine by the action of a CoA-ligase (Bell & Turner, 1976a;McGilvray & Morris, 1969). L-Threonine aldolase (EC 2.1.2.1)-initiated dissimilation has been demonstrated in a limited number of bacteria (Morris, 1969;Willetts & Turner, 1971 ;Kumagai et al, 1972).…”

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Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine hydro-lyase (deaminating) in a species of Corynebacterium

Bell

Turner

1977

Biochemical Journal

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1. Three bacterial isolates capable of growth on L-threonine medium only when supplemented with branched-chain amino acids, and possessing high L-threonine dehydratase activity, were examined to elucidate the catabolic route for the amino acid. 2. Growth, manometric, radiotracer and enzymic experiments indicated that L-threonine was catabolized by initial deamination to 2-oxobutyrate and thence to propionate. No evidence was obtained for the involvement of L-threonine 3-dehydrogenase or L-threonine aldolase in threonine catabolism. 3. L-Threonine dehydratase of Corynebacterium sp. F5 (N.C.I.B. 11102) was partially purified and its kinetic properties were examined. The enzyme exhibited a sigmoid kinetic response to substrate concentration. The concentration of substrate giving half the maximum velocity, [SO.5], was 40mM and the Hill coefficient (h) was 2.0. L-Isoleucine inhibited enzyme activity markedly, causing 50% inhibition at 60,UM, but did not affect the Hill constant. At the fixed L-threonine concentration of 10mM, the effect of L-valine was biphasic, progressive activation occurring at concentrations up to 2mM-L-valine, but was abolished by higher concentrations. Substrate-saturation plots for the L-valine-activated enzyme exhibited normal MichaelisMenten kinetics with a Hill coefficient (h) of 1.0. The kinetic properties of the enzyme were thus similar to those of the 'biosynthetic' isoenzyme from Rhodopseudomonas spheroides rather than those of the enteric bacteria. 4. The synthesis of L-threonine dehydratase was constitutive and was not subject to multivalent repression by L-isoleucine or other branched-chain amino acids either singly or in combination. 5. The catabolism of L-threonine, apparently initiated by a 'biosynthetic' L-threonine dehydratase in the isolates studied, depended on the concomitant catabolism of branched-chain amino acids. The biochemical basis of this dependence appeared to lie in the further catabolism of 2-oxobutyrate by enzymes which required branched-chain 2-oxo acids for their induction.

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Discovery of potent polyphosphate kinase 1 (PPK1) inhibitors using structure‐based exploration of PPK1Pharmacophoric space coupled with docking analyses

Bashatwah

Khanfar

Bardaweel

2018

J of Molecular Recognition

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Inorganic polyphosphate (polyP) is present in all living forms of life. Studied mainly in prokaryotes, polyP and its associated enzymes are vital in diverse metabolic activities, in some structural functions, and most importantly in stress responses. Bacterial species, including many pathogens, encode a homolog of a major polyP synthesis enzyme, Poly Phosphate Kinase (PPK) with 2 different genes coding for PPK1 and PPK2. Genetic deletion of the ppk1 gene leads to reduced polyP levels and the consequent loss of virulence and stress adaptation responses. This far, no PPK1 homolog has been identified in higher-order eukaryotes, and, therefore, PPK1 represents a novel target for chemotherapy. The aim of the current study is to investigate PPK1 from Escherichia coli with comprehensive understanding of the enzyme's structure and binding sites, which were used to design pharmacophores and screen a library of compounds for potential discovery of selective PPK1 inhibitors. Verification of the resultant inhibitors activities was conducted using a combination of mutagenic and chemical biological approaches. The metabolic phenotypic maps of the wild type E. coli (WT) and ppk1 knockout mutant were generated and compared with the metabolic map of the chemically inhibited WT. In addition, biofilm formation ability was measured in WT, ppk1 knockout mutant, and the chemically inhibited WT. The results demonstrated that chemical inhibition of PPK1, with the designed inhibitors, was equivalent to gene deletion in altering specific metabolic pathways, changing the metabolic fingerprint, and suppressing the ability of E. coli to form a biofilm.

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Bacterial catabolism of threonine. Threonine degradation initiated by L-threonine-NAD+ oxidoreductase

Cited by 44 publications

References 32 publications

Identification of glyA (Encoding Serine Hydroxymethyltransferase) and Its Use Together with the Exporter ThrE To Increase l -Threonine Accumulation by Corynebacterium glutamicum

Identification of glyA (Encoding Serine Hydroxymethyltransferase) and Its Use Together with the Exporter ThrE To Increase l -Threonine Accumulation by Corynebacterium glutamicum

Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine hydro-lyase (deaminating) in a species of Corynebacterium

Discovery of potent polyphosphate kinase 1 (PPK1) inhibitors using structure‐based exploration of PPK1Pharmacophoric space coupled with docking analyses

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