The C-branched pentose sugar d-apiose is important for plant cell wall development. Its biosynthesis as urdine diphosphate-d-apiose (UDP-d-apiose) involves decarboxylation of the UDP-d-glucuronic acid precursor coupled with pyranosyl-to-furanosyl sugar ring contraction. This unusual multistep reaction is catalysed in a single active site by UDP-d-apiose/UDP-d-xylose synthase (UAXS). Here we decipher the UAXS catalytic mechanism on the basis of crystal structures of the enzyme (which is from Arabidopsis thaliana), molecular dynamics simulations that are expanded by hybrid quantum mechanics/molecular mechanics calculations and mutational mechanistic analyses. Our studies show how UAXS uniquely integrates a classical catalytic cycle of oxidation and reduction by a tightly bound nicotinamide co-enzyme with retroaldol/aldol chemistry for the sugar ring contraction. We further demonstrate that decarboxylation occurs only after the sugar ring opening and identify that the thiol group of Cys100 steers the sugar skeleton rearrangement by proton transfer to and from C3′. The mechanistic features of UAXS highlight the evolutionary expansion of the basic catalytic apparatus of short-chain dehydrogenases/reductases for functional versatility in sugar biosynthesis.
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The contributions of P. Scudieri (enzyme characterization), A. Lepak (UDP-apiose HPLC assay) and J. Coines (analysis of MD trajectories) are gratefully acknowledged. This work was supported by the Federal Ministry of Science, Research and Economy (BMWFW), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol, and the Government of Lower Austrian and Business Agency Vienna through the COMET-funding programme managed by the Austrian Research Promotion Agency FFG. Funding from the Austrian Science Funds (FWF; I-3247 to B.N. and A.J.E.B.) and by the Italian Ministry of Education, University and Research (MIUR) (Dipartimenti di Eccellenza Program (2018–2022)—Department of Biology and Biotechnology ‘L. Spallanzani’ University of Pavia) is acknowledged.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Methods, Table 1, Figs. 1–47 and references
Motions of the UAXS active site during MD simulations (replica 1)
Motions of the UAXS active site during MD simulations (replica 2)
Initial coordinates for replica 1
Initial coordinates for replica 2
Final coordinates after 300 ns of MD simulations for replica 1
Final coordinates after 300 ns of MD simulations for replica 2
QM/MM optimized geometry of the QM region of the QM/MM calculation in the XYZ file format for a snapshot taken from the MD of replica 1
QM/MM optimized geometry of the QM region of the QM/MM calculation in the XYZ file format, the snapshot is taken from the MD for replica 2
Snapshot from replica 1 used for QM/MM calculations, it represents the Michaelis complex before QM/MM optimization
Snapshot from replica 2 used for QM/MM calculations, it represents the complex before QM/MM optimization
QM/MM optimized geometry of the entire complex including the QM and MM region from the QM/MM calculation in the XYZ file format, the snapshot was taken from the MD of replica 1.
QM/MM optimized geometry of the entire complex including the QM and MM region of the QM/MM calculation in the XYZ file format, the snapshot was taken from the MD of replica 2
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Savino, S., Borg, A.J.E., Dennig, A. et al. Deciphering the enzymatic mechanism of sugar ring contraction in UDP-apiose biosynthesis. Nat Catal 2, 1115–1123 (2019). https://doi.org/10.1038/s41929-019-0382-8