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Pyruvate:quinone oxidoreductase in Corynebacterium glutamicum: molecular analysis of the pqo gene, significance of the enzyme, and phylogenetic aspects.

ABSTRACT: Corynebacterium glutamicum recently has been shown to possess pyruvate:quinone oxidoreductase (PQO), catalyzing the oxidative decarboxylation of pyruvate to acetate and CO2 with a quinone as the electron acceptor. Here, we analyze the expression of the C. glutamicum pqo gene, investigate the relevance of the PQO enzyme for growth and amino acid production, and perform phylogenetic studies. Expression analyses revealed that transcription of pqo is initiated 45 bp upstream of the translational start site and that it is organized in an operon together with genes encoding a putative metal-activated pyridoxal enzyme and a putative activator protein. Inactivation of the chromosomal pqo gene led to the absence of PQO activity; however, growth and amino acid production were not affected under either condition tested. Introduction of plasmid-bound pqo into a pyruvate dehydrogenase complex-negative C. glutamicum strain partially relieved the growth phenotype of this mutant, indicating that high PQO activity can compensate for the function of the pyruvate dehydrogenase complex. To investigate the distribution of PQO enzymes in prokaryotes and to clarify the relationship between PQO, pyruvate oxidase (POX), and acetohydroxy acid synthase enzymes, we compiled and analyzed the phylogeny of respective proteins deposited in public databases. The analyses revealed a wide distribution of PQOs among prokaryotes, corroborated the hypothesis of a common ancestry of the three enzymes, and led us to propose that the POX enzymes of Lactobacillales were derived from a PQO.

SUBMITTER: Schreiner ME

PROVIDER: S-EPMC1367228 | biostudies-literature | 2006 Feb

REPOSITORIES: biostudies-literature

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Pyruvate:quinone oxidoreductase in Corynebacterium glutamicum: molecular analysis of the pqo gene, significance of the enzyme, and phylogenetic aspects.

Schreiner Mark E ME Riedel Christian C Holátko Jiri J Pátek Miroslav M Eikmanns Bernhard J BJ

Journal of bacteriology 20060201 4

Corynebacterium glutamicum recently has been shown to possess pyruvate:quinone oxidoreductase (PQO), catalyzing the oxidative decarboxylation of pyruvate to acetate and CO2 with a quinone as the electron acceptor. Here, we analyze the expression of the C. glutamicum pqo gene, investigate the relevance of the PQO enzyme for growth and amino acid production, and perform phylogenetic studies. Expression analyses revealed that transcription of pqo is initiated 45 bp upstream of the translational sta ...[more]

PMID: 16452416

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Project description:Pyruvate kinase (Pyk) catalyzes the generation of pyruvate and ATP in glycolysis and functions as a key switch in the regulation of carbon flux distribution. Both the substrates and products of Pyk are involved in the tricarboxylic acid cycle, anaplerosis and energy anabolism, which places Pyk at a primary metabolic intersection. Pyks are highly conserved in most bacteria and lower eukaryotes. Corynebacterium glutamicum is an industrial workhorse for the production of various amino acids and organic acids. Although C. glutamicum was assumed to possess only one Pyk (pyk1, NCgl2008), NCgl2809 was annotated as a pyruvate kinase with an unknown role.Here, we identified that NCgl2809 was a novel pyruvate kinase (pyk2) in C. glutamicum. Complementation of the WT?pyk1?pyk2 strain with the pyk2 gene restored its growth on D-ribose, which demonstrated that Pyk2 could substitute for Pyk1 in vivo. Pyk2 was co-dependent on Mn2+ and K+ and had a higher affinity for ADP than phosphoenolpyruvate (PEP). The catalytic activity of Pyk2 was allosterically regulated by fructose 1,6-bisphosphate (FBP) activation and ATP inhibition. Furthermore, pyk2 and ldhA, which encodes L-lactate dehydrogenase, were co-transcribed as a bicistronic mRNA under aerobic conditions and pyk2 deficiency had a slight effect on the intracellular activity of Pyk. However, the mRNA level of pyk2 in the wild-type strain under oxygen deprivation was 14.24-fold higher than that under aerobic conditions. Under oxygen deprivation, pyk1 or pyk2 deficiency decreased the generation of lactic acid, and the overexpression of either pyk1 or pyk2 increased the production of lactic acid as the activity of Pyk increased. Fed-batch fermentation of the pyk2-overexpressing WT?pyk1 strain produced 60.27?±?1.40 g/L of lactic acid, which was a 47% increase compared to the parent strain under oxygen deprivation.Pyk2 functioned as a pyruvate kinase and contributed to the increased level of Pyk activity under oxygen deprivation.

Dataset Information

Pyruvate:quinone oxidoreductase in Corynebacterium glutamicum: molecular analysis of the pqo gene, significance of the enzyme, and phylogenetic aspects.

Publications

Pyruvate:quinone oxidoreductase in Corynebacterium glutamicum: molecular analysis of the pqo gene, significance of the enzyme, and phylogenetic aspects.

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