Dataset Information

Metabolism of ?-valine via a CoA-dependent ammonia lyase pathway.

ABSTRACT: Pseudomonas species strain SBV1 can rapidly grow on medium containing ?-valine as a sole nitrogen source. The tertiary amine feature of ?-valine prevents direct deamination reactions catalyzed by aminotransferases, amino acid dehydrogenases, and amino acid oxidases. However, lyase- or aminomutase-mediated conversions would be possible. To identify enzymes involved in the degradation of ?-valine, a PsSBV1 gene library was prepared and used to complement the ?-valine growth deficiency of a closely related Pseudomonas strain. This resulted in the identification of a gene encoding ?-valinyl-coenzyme A ligase (BvaA) and two genes encoding ?-valinyl-CoA ammonia lyases (BvaB1 and BvaB2). The BvaA protein demonstrated high sequence identity to several known phenylacetate CoA ligases. Purified BvaA enzyme did not convert phenyl acetic acid but was able to activate ?-valine in an adenosine triphosphate (ATP)- and CoA-dependent manner. The substrate range of the enzyme appears to be narrow, converting only ?-valine and to a lesser extent, 3-aminobutyrate and ?-alanine. Characterization of BvaB1 and BvaB2 revealed that both enzymes were able to deaminate ?-valinyl-CoA to produce 3-methylcrotonyl-CoA, a common intermediate in the leucine degradation pathway. Interestingly, BvaB1 and BvaB2 demonstrated no significant sequence identity to known CoA-dependent ammonia lyases, suggesting they belong to a new family of enzymes. BLAST searches revealed that BvaB1 and BvaB2 show high sequence identity to each other and to several enoyl-CoA hydratases, a class of enzymes that catalyze a similar reaction with water instead of amine as the leaving group.

SUBMITTER: Otzen M

PROVIDER: S-EPMC4619459 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Metabolism of β-valine via a CoA-dependent ammonia lyase pathway.

Otzen Marleen M Crismaru Ciprian G CG Postema Christiaan P CP Wijma Hein J HJ Heberling Matthew M MM Szymanski Wiktor W de Wildeman Stefaan S Janssen Dick B DB

Applied microbiology and biotechnology 20150526 21

Pseudomonas species strain SBV1 can rapidly grow on medium containing β-valine as a sole nitrogen source. The tertiary amine feature of β-valine prevents direct deamination reactions catalyzed by aminotransferases, amino acid dehydrogenases, and amino acid oxidases. However, lyase- or aminomutase-mediated conversions would be possible. To identify enzymes involved in the degradation of β-valine, a PsSBV1 gene library was prepared and used to complement the β-valine growth deficiency of a closely ...[more]

PMID: 26004802

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Project description:Benzenoid-derived metabolites act as precursors for a wide variety of products involved in essential metabolic roles in eukaryotic cells. They are synthesized in plants and some fungi through the phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) pathways. Ascomycete yeasts and animals both lack the capacity for PAL/TAL pathways, and metabolic reactions leading to benzenoid synthesis in these organisms have remained incompletely known for decades. Here, we show genomic, transcriptomic, and metabolomic evidence that yeasts use a mandelate pathway to synthesize benzenoids, with some similarities to pathways used by bacteria. We conducted feeding experiments using a synthetic fermentation medium that contained either 13C-phenylalanine or 13C-tyrosine, and, using methylbenzoylphosphonate (MBP) to inhibit benzoylformate decarboxylase, we were able to accumulate intracellular intermediates in the yeast Hanseniaspora vineae To further confirm this pathway, we tested in separate fermentation experiments three mutants with deletions in the key genes putatively proposed to form benzenoids (Saccharomyces cerevisiae aro10?, dld1?, and dld2? strains). Our results elucidate the mechanism of benzenoid synthesis in yeast through phenylpyruvate linked with the mandelate pathway to produce benzyl alcohol and 4-hydroxybenzaldehyde from the aromatic amino acids phenylalanine and tyrosine, as well as sugars. These results provide an explanation for the origin of the benzoquinone ring, 4-hydroxybenzoate, and suggest that Aro10p has benzoylformate and 4-hydroxybenzoylformate decarboxylase functions in yeast.IMPORTANCE We present here evidence of the existence of the mandelate pathway in yeast for the synthesis of benzenoids. The link between phenylpyruvate- and 4-hydroxyphenlypyruvate-derived compounds with the corresponding synthesis of benzaldehydes through benzoylformate decarboxylation is demonstrated. Hanseniaspora vineae was used in these studies because of its capacity to produce benzenoid derivatives at a level 2 orders of magnitude higher than that produced by Saccharomyces Contrary to what was hypothesized, neither ?-oxidation derivatives nor 4-coumaric acid is an intermediate in the synthesis of yeast benzenoids. Our results might offer an answer to the long-standing question of the origin of 4-hydroxybenzoate for the synthesis of Q10 in humans.

Dataset Information

Metabolism of ?-valine via a CoA-dependent ammonia lyase pathway.

Publications

Metabolism of β-valine via a CoA-dependent ammonia lyase pathway.

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