Isotope Probing of the UDP-Apiose/UDP-Xylose Synthase Reaction: Evidence of a Mechanism via a Coupled Oxidation and Aldol Cleavage

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-alpha-D-glucuronic acid (UDP-GlcA) to the Api precursor UDP-alpha-D-apiose (UDP-Api). We examined the mechanism of UDP-Api/UDP-alpha-D-xylose synthase (UAXS) with site-selectively H-2-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.