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Characterization of an L-Ascorbate Catabolic Pathway with Unprecedented Enzymatic Transformations

submitted on 11.09.2019, 21:50 and posted on 12.09.2019, 16:59 by Tyler Stack, Katelyn Morrison, Thomas Dettmer, Brendan Wille, Chan Kim, Ryan Joyce, Madison Jermain, Yadanar Than Naing, Khadija Bhatti, Brian San Francisco, Michael S. Carter, John A. Gerlt

L-Ascorbate (vitamin C) is ubiquitous in both our diet and the environment. Ralstonia eutropha H16 (Cupriavidus necator ATCC 17699) uses L-ascorbate as sole carbon source but lacks the genes encoding the known catabolic pathways. RNAseq identified eight candidate catabolic genes. Sequence similarity networks and genome neighborhood networks guided predictions for function of the encoded proteins; 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 b561 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 alpha-ketoglutarate by a previously characterized pathway.




Email Address of Submitting Author


University of Illinois – Urbana/Champaign


United States of America

ORCID For Submitting Author


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No conflict of interest


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in Journal of the American Chemical Society