Mechanistic characterization of UDP‐glucuronic acid 4‐epimerase

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 AE 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 (AE0.1) 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.

Expanding the Enzyme Repertoire for Sugar Nucleotide Epimerization: the CDP-Tyvelose 2-Epimerase from Thermodesulfatator atlanticus for Glucose/Mannose Interconversion

“…S5). High-performance liquid chromatography (HPLC) analysis ( 21 ) revealed NAD (not NADP) to be the cofactor, being present predominantly in the oxidized form (Fig. S6) (NAD + , 98.1% ± 0.4%; NADH, 1.9% ± 0.4%).…”

“…The epimerization reaction appears to involve complex bond rotations within the substrate’s pyrophosphate group to enable flipped positions of the transient 2-keto intermediate for nonstereospecific reduction. Catalytic oxidation-reduction coordinated with molecular rotation in the enzyme’s active site is a general mechanistic principle of sugar nucleotide-modifying epimerases of the SDR superfamily ( 10 , 19 , 21 , 58 , 59 ).…”

“…However, the type of catalysis used by these epimerases, effectively a combination of concerted general acid/base catalysis, limits the scope of transformations available to plain monosaccharides (13,16). Epimerases acting on sugar nucleotide substrates offer a richer portfolio of catalytic chemistry, including oxidation/reduction and elimination/re-addition (17)(18)(19)(20)(21)(22). They can thus expand the range of substrate transformations accessible for epimerization (10).…”

“…Sugar nucleotide-dependent epimerases hence complement the synthetic repertoire and the applied perspective of enzymatic epimerization. However, limitations in their applicability to carbohydrate synthesis can arise from the high substrate specificity that many of these enzymes exhibit ( 19 , 21 , 26 ). Novel epimerases more promiscuous in their acceptance of sugar nucleotide substrates are hence of high interest.…”

Overdominance at the Gene Expression Level Plays a Critical Role in the Hybrid Root Growth of Brassica napus

“…AGAL1 has been discovered in quiescent center (QC) cells from the meristem of the roots [87,88]. The enzyme UDP-glucuronate 4-epimerase (GALE) catalyzes UDP-glucose into active products that are involved in many other metabolic pathways, including glucuronate and pentose interconversions and nucleotide sugar metabolism [89][90][91].…”