TY - JOUR
T1 - Characterization of an l -Ascorbate Catabolic Pathway with Unprecedented Enzymatic Transformations
AU - Stack, Tyler M.M.
AU - Morrison, Katelyn N.
AU - Dettmer, Thomas M.
AU - Wille, Brendan
AU - Kim, Chan
AU - Joyce, Ryan
AU - Jermain, Madison
AU - Naing, Yadanar Than
AU - Bhatti, Khadija
AU - Francisco, Brian San
AU - Carter, Michael S.
AU - Gerlt, John A.
N1 - This research was supported by the National Institutes of Health P01GM118303, Drury University, and Salisbury University. We thank Dr. Xudong Guan, NMR specialist at the Institute for Genomic Biology, University of Illinois at Urbana–Champaign, for assistance with the acquisition of NMR spectra. The NMR data were collected in the IGB Core on a 600 MHz NMR funded by NIH grant number S10-RR028833. We thank Dr. Zhong Lucas Li, Director of Metabolomics at the Roy J. Carver Biotechnology Center, for collecting the mass spectral data. We thank the Drury University 2016 Fall BIOL 322 Advanced Genetics course for their efforts in primer design and molecular cloning.
PY - 2020/1/29
Y1 - 2020/1/29
N2 - 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 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 α-ketoglutarate by a previously characterized pathway. The pathway is found in hundreds of bacteria, including the pathogens Pseudomonas aeruginosa and Acinetobacter baumannii.
AB - 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 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 α-ketoglutarate by a previously characterized pathway. The pathway is found in hundreds of bacteria, including the pathogens Pseudomonas aeruginosa and Acinetobacter baumannii.
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U2 - 10.1021/jacs.9b09863
DO - 10.1021/jacs.9b09863
M3 - Article
C2 - 31917558
AN - SCOPUS:85078535490
SN - 0002-7863
VL - 142
SP - 1657
EP - 1661
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 4
ER -