Mechanistic characterization of UDP‐glucuronic acid 4‐epimerase. (5th August 2020)

• Record Type: Journal Article
• Authors: Borg, Annika J. E.; Dennig, Alexander; Weber, Hansjörg; Nidetzky, Bernd
• Is Part Of: FEBS journal. Volume 288:Number 4(2021)
• Page Start: 1163
• Page End: 1178
• Publication Date: 2020-08-05
• Subjects: decarboxylase -- epimerase -- kinetic isotope effect -- short‐chain dehydrogenase/reductase -- UDP‐glucuronic acid; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.15478
• Languages: English
• ISSNs: 1742-464X

Abstract

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 ( k cat = 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 k cat 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. Abstract : UDP‐glucuronic acid 4‐epimerase is a short‐chain dehydrogenase/reductase that uses tightly bound NAD + to catalyze interconversion of UDP‐glucuronic acid and UDP‐galacturonic acid. Here, the authors present a biochemical mechanistic study of the enzyme from Bacillus cereus . Epimerization involves oxidation at the substrate C4 and nonstereoselective reduction in a 4‐keto intermediate. Substrate positioning in a kinetically slow binding step can establish stereo‐electronic constraints to prevent decarboxylation of the intermediate.