Mechanistic characterization of UDP-glucuronic acid 4-epimerase

Annika J.E. Borg, Alexander Dennig, Hansjörg Weber, Bernd Nidetzky*

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

UDP-glucuronic acid (UDP-GlcA) is a central precursor in sugar nucleotide biosynthesis and common substrate for C4-epimerases and decarboxylases releasing UDP-galacturonic acid (UDP-GalA) and UDP-pentose products, respectively. Despite the different reactions catalyzed, the enzymes are believed to share mechanistic analogy rooted in their joint membership to the short-chain dehydrogenase/reductase (SDR) protein superfamily: Oxidation at the substrate C4 by enzyme-bound NAD+ initiates the catalytic pathway. Here, we present mechanistic characterization of the C4-epimerization of UDP-GlcA, which in comparison with the corresponding decarboxylation has been largely unexplored. The UDP-GlcA 4-epimerase from Bacillus cereus functions as a homodimer and contains one NAD+/subunit (kcat = 0.25 ± 0.01 s−1). The epimerization of UDP-GlcA proceeds via hydride transfer from and to the substrate’s C4 while retaining the enzyme-bound cofactor in its oxidized form (≥ 97%) at steady state and without trace of decarboxylation. The kcat for UDP-GlcA conversion shows a kinetic isotope effect of 2.0 (±0.1) derived from substrate deuteration at C4. The proposed enzymatic mechanism involves a transient UDP-4-keto-hexose-uronic acid intermediate whose formation is rate-limiting overall, and is governed by a conformational step before hydride abstraction from UDP-GlcA. Precise positioning of the substrate in a kinetically slow binding step may be important for the epimerase to establish stereo-electronic constraints in which decarboxylation of the labile β-keto acid species is prevented effectively. Mutagenesis and pH studies implicate the conserved Tyr149 as the catalytic base for substrate oxidation and show its involvement in the substrate positioning step. Collectively, this study suggests that based on overall mechanistic analogy, stereo-electronic control may be a distinguishing feature of catalysis by SDR-type epimerases and decarboxylases active on UDP-GlcA.

Original languageEnglish
Pages (from-to)1163-1178
Number of pages16
JournalThe FEBS Journal
Volume288
Issue number4
Early online date9 Jul 2020
DOIs
Publication statusPublished - Feb 2021

Keywords

  • decarboxylase
  • epimerase
  • kinetic isotope effect
  • short-chain dehydrogenase/reductase
  • UDP-glucuronic acid

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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