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‐selectively 2H‐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.

Release, Recycle, Rebuild: Cell-Wall Remodeling, Autodegradation, and Sugar Salvage for New Wall Biosynthesis during Plant Development

“…For example, 2-fluoro-fucose inhibits the incorporation of fucose into Arabidopsis cell walls (Dumont et al, 2015;Villalobos et al, 2015). NDP-sugar interconversion has also been investigated using fluorinated compounds as described in studies using synthesized UDP-2-fluoro-glucuronic acid to monitor the conversion and mechanism of UDP-apiose formation by UDP-D-apiose/UDP-D-xylose synthase (Choi et al, 2011;Eixelberger et al, 2017). In maize and pea etiolated seedlings, 2-deoxyglucose inhibits callose production during gravitropic responses (Jaffe and Leopold, 1984).…”

Deciphering the enzymatic mechanism of sugar ring contraction in UDP-apiose biosynthesis

“…It involves decarboxylation coupled to sugar ring contraction to generate a five-membered ring furanosyl product. [11][12][13] The enzyme also produces UDPxylose, a six-membered ring UDP-xylopyranose (3), in consistent ratios, typically about 1:1 at neutral pH. 6,13 A plausible reaction path has been proposed based on mechanistic studies spanning several decades, as shown in Figure 1.…”

“…[11][12][13] The enzyme also produces UDPxylose, a six-membered ring UDP-xylopyranose (3), in consistent ratios, typically about 1:1 at neutral pH. 6,13 A plausible reaction path has been proposed based on mechanistic studies spanning several decades, as shown in Figure 1. 6,[9][10][11][12][13][14][15][16][17][18][19] The relative timing of the catalytic steps has been recently elucidated from kinetic isotope effects on the enzymatic rates as well as on the distribution of UDP-apiose (1a) and UDP-xylose (3).…”

“…6,[9][10][11][12][13][14][15][16][17][18][19] The relative timing of the catalytic steps has been recently elucidated from kinetic isotope effects on the enzymatic rates as well as on the distribution of UDP-apiose (1a) and UDP-xylose (3). 13 UDP-GlcA is first oxidized at C4' by the tightly bound NAD + . Subsequently, deprotonation of the 2'-OH facilitates a C2'-C3' aldol cleavage.…”

“…Subsequently, deprotonation of the 2'-OH facilitates a C2'-C3' aldol cleavage. [11][12][13] Decarboxylation thus occurs on the ring-opened oxidized substrate. 13 Rearrangement of the carbon skeleton yields either UDPapiose aldehyde (4) or UDP-4-keto-xylose (5) whose reduction by NADH gives the respective furanosyl or pyranosyl products.…”