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CDNA Isolation and Functional Characterization of UDP-d-glucuronic Acid 4-Epimerase Family from Ornithogalum caudatum.

ABSTRACT: d-Galacturonic acid (GalA) is an important component of GalA-containing polysaccharides in Ornithogalum caudatum. The incorporation of GalA into these polysaccharides from UDP-d-galacturonic acid (UDP-GalA) was reasonably known. However, the cDNAs involved in the biosynthesis of UDP-GalA were still unknown. In the present investigation, one candidate UDP-d-glucuronic acid 4-epimerase (UGlcAE) family with three members was isolated from O. caudatum based on RNA-Seq data. Bioinformatics analyses indicated all of the three isoforms, designated as OcUGlcAE1~3, were members of short-chain dehydrogenases/reductases (SDRs) and shared two conserved motifs. The three full-length cDNAs were then transformed to Pichia pastoris GS115 for heterologous expression. Data revealed both the supernatant and microsomal fractions from the recombinant P. pastoris expressing OcUGlcAE3 can interconvert UDP-GalA and UDP-d-glucuronic acid (UDP-GlcA), while the other two OcUGlcAEs had no activity on UDP-GlcA and UDP-GalA. Furthermore, expression analyses of the three epimerases in varied tissues of O. caudatum were performed by real-time quantitative PCR (RT-qPCR). Results indicated OcUGlcAE3, together with the other two OcUGlcAE-like genes, was root-specific, displaying highest expression in roots. OcUGlcAE3 was UDP-d-glucuronic acid 4-epimerase and thus deemed to be involved in the biosynthesis of root polysaccharides. Moreover, OcUGlcAE3 was proposed to be environmentally induced.

SUBMITTER: Yin S

PROVIDER: S-EPMC6273887 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:The UDP-glucuronosyltransferase (UGT) isozyme system is critical for protecting the body against endogenous and exogenous chemicals by linking glucuronic acid donated by UDP-glucuronic acid to a lipophilic acceptor substrate. UGTs convert metabolites, dietary constituents, and environmental toxicants to highly excretable glucuronides. Because of difficulties associated with purifying endoplasmic reticulum-bound UGTs for structural studies, we carried out homology-based computer modeling to aid analysis. The search found structural homology in Escherichia coli UDP-galactose 4-epimerase. Consistent with predicted similarities involving the common UDP moiety in substrate/inhibitor, UDP-glucose and UDP-hexanol amine caused competitive inhibition by Lineweaver-Burk plots. Among predicted binding sites N292, K314, K315, and K404 in UGT1A10, two informative sets of mutants K314R/Q/A/E/G and K404R/E had null activities or 2.7-fold higher/50% less activity, respectively. Scatchard analysis of binding data of the affinity ligand, 5-azidouridine-[beta- (32)P]diphosphoglucuronic acid, to purified UGT1A10-His or UGT1A7-His revealed high- and low-affinity binding sites. 2-Nitro-5-thiocyanobenzoic acid-digested UGT1A10-His bound with the radiolabeled affinity ligand revealed an 11.3 and 14.3 kDa peptide associated with K314 and K404, respectively, in a discontinuous SDS-PAGE system. Similar treatment of 1A10His-K314A bound with the ligand lacked both peptides; 1A10-HisK404R- and 1A10-HisK404E showed 1.3-fold greater and 50% less label in the 14.3 kDa peptide, respectively, compared to 1A10-His without affecting the 11.3 kDa peptide. Scatchard analysis of binding data of the affinity ligand to 1A10His-K404R and -K404E showed a 6-fold reduction and a large increase in K d, respectively. Our results indicate that K314 and K404 are required UDP-glcA binding sites in 1A10, that K404 controls activity and high-affinity sites, and that K314 and K404 are strictly conserved in 70 aligned UGTs, except for S321, equivalent to K314, in UGT2B15 and 2B17 and I321 in the inactive UGT8, which suggests UGT2B15 and 2B17 contain suboptimal activity. Hence our data strongly support UDP-glcA binding to K314 and K404 in UGT1A10.

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CDNA Isolation and Functional Characterization of UDP-d-glucuronic Acid 4-Epimerase Family from Ornithogalum caudatum.

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