Characterization of an l‑Ascorbate Catabolic Pathway with Unprecedented Enzymatic Transformations

l-Ascorbate (vitamin C) is ubiquitous in both our diet and the environment. Here we report that Ralstonia eutropha H16 (Cupriavidus necator ATCC 17699) uses l-ascorbate as sole carbon source via a novel catabolic pathway. RNaseq identified eight candidate catabolic genes, sequence similarity network...

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Bibliographic Details
Published in:	Journal of the American Chemical Society Vol. 142; no. 4; pp. 1657 - 1661
Main Authors:	Stack, Tyler M. M, Morrison, Katelyn N, Dettmer, Thomas M, Wille, Brendan, Kim, Chan, Joyce, Ryan, Jermain, Madison, Naing, Yadanar Than, Bhatti, Khadija, Francisco, Brian San, Carter, Michael S, Gerlt, John A
Format:	Journal Article
Language:	English
Published:	United States American Chemical Society 29-01-2020
Subjects:	Ascorbic Acid - metabolism Bacterial Proteins - metabolism Cupriavidus necator - enzymology Cupriavidus necator - genetics Cupriavidus necator - metabolism Enzymes - metabolism Genes, Bacterial Oxidation-Reduction
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Summary:	l-Ascorbate (vitamin C) is ubiquitous in both our diet and the environment. Here we report that Ralstonia eutropha H16 (Cupriavidus necator ATCC 17699) uses l-ascorbate as sole carbon source via a novel catabolic pathway. RNaseq identified eight candidate catabolic genes, sequence similarity networks, and genome neighborhood networks guided predictions for function of the encoded proteins, and the predictions were confirmed by in vitro assays and in vivo growth phenotypes of gene deletion mutants. l-Ascorbate, a lactone, is oxidized and ring-opened by enzymes in the cytochrome b 561 and gluconolactonase families, respectively, to form 2,3-diketo-l-gulonate. A protein predicted to have a WD40-like fold catalyzes an unprecedented benzilic acid rearrangement involving migration of a carboxylate group to form 2-carboxy-l-lyxonolactone; the lactone is hydrolyzed by a member of the amidohydrolase superfamily to yield 2-carboxy-l-lyxonate. A member of the PdxA family of oxidative decarboxylases catalyzes a novel decarboxylation that uses NAD+ catalytically. The product, l-lyxonate, is catabolized to α-ketoglutarate by a previously characterized pathway. The pathway is found in hundreds of bacteria, including the pathogens Pseudomonas aeruginosa and Acinetobacter baumannii.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Present Addresses Department of Biological Sciences, Salisbury University, Salisbury, Maryland 21801, United States
ISSN:	0002-7863 1520-5126
DOI:	10.1021/jacs.9b09863