title: Isotope Probing of the UDP-Apiose/UDP-Xylose Synthase Reaction: Evidence of a Mechanism via a Coupled Oxidation and Aldol Cleavage.
abstract: The C-branched sugar d-apiose (Api) is essential for plant cell-wall development. An enzyme-catalyzed decarboxylation/pyranoside ring-contraction reaction leads from UDP-α-d-glucuronic acid (UDP-GlcA) to the Api precursor UDP-α-d-apiose (UDP-Api). We examined the mechanism of UDP-Api/UDP-α-d-xylose synthase (UAXS) with site-selectively2H-labeled and deoxygenated substrates. The analogue UDP-2-deoxy-GlcA, which prevents C-2/C-3 aldol cleavage as the plausible initiating step of pyranoside-to-furanoside conversion, did not give the corresponding Api product. Kinetic isotope effects (KIEs) support an UAXS mechanism in which substrate oxidation by enzyme-NAD+and retro-aldol sugar ring-opening occur coupled in a single rate-limiting step leading to decarboxylation. Rearrangement and ring-contracting aldol addition in an open-chain intermediate then give the UDP-Api aldehyde, which is intercepted via reduction by enzyme-NADH.
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, 8010, Graz, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Eixelsberger
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, 8010, Graz, Austria.
authors.forename: Doroteja
authors.initials: D
authors.lastname: Horvat
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, 8010, Graz, Austria.
authors.forename: Alexander
authors.initials: A
authors.lastname: Gutmann
authors.affiliation: Institute of Organic Chemistry, Graz University of Technology, NAWI Graz, Stremayrgasse 9, 8010, Graz, Austria.
authors.forename: Hansjörg
authors.initials: H
authors.lastname: Weber
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, 8010, Graz, Austria.|Austrian Centre of Industrial Biotechnology (acib), Petersgasse 14, 8010, Graz, Austria.
authors.forename: Bernd
authors.identifier: 0000-0002-5030-2643
authors.initials: B
authors.lastname: Nidetzky
keywords: aldol reactions; carbohydrates; enzyme catalysis; reaction mechanism; ring contraction