Uronate isomerase, a member of the amidohydrolase superfamily, catalyzes the isomerization of D-glucuronate and D-fructuronate. During the interconversion of substrate and product the hydrogen at C2 of D-glucuronate is transferred to the pro-R position at C1 of the product, D-fructuronate. The exchange of the transferred hydrogen with solvent deuterium occurs at a rate that is 4 orders of magnitude slower than the interconversion of substrate and product. The enzyme catalyzes the elimination of fluoride from 3-deoxy-3-fluoro-D-glucuronate. These results have been interpreted to suggest a chemical reaction mechanism in which an active site base abstracts the proton from C2 of D-glucuronate to form a cis-enediol intermediate. The conjugate acid then transfers this proton to C1 of the cis-enediol intermediate to form D-fructuronate. The loss of fluoride from 3-deoxy-3-fluoro-D-glucuronate is consistent with a stabilized carbanion at C2 of the substrate during substrate turnover. The slow exchange of the transferred hydrogen with solvent water is consistent with a shielded conjugate acid after abstraction of the proton from either D-glucuronate or D-fructuronate during the isomerization reaction. This conclusion is supported by the competitive inhibition of the enzymatic reaction by D-arabinaric acid and the monohydroxamate derivative with Ki values of 13 and 670 nM, respectively. There is no evidence to support a hydride transfer mechanism for uronate isomerase. The wild type enzyme was found to contain 1 equiv of zinc per subunit. The divalent cation could be removed by dialysis against the metal chelator, dipicolinate. However, the apoenzyme has the same catalytic activity as the Zn-substituted enzyme and thus the divalent metal ion is not required for enzymatic activity. This is the only documented example of a member in the amidohydrolase superfamily that does not require one or two divalent cations for enzymatic activity.
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http://dx.doi.org/10.1021/bi060531l | DOI Listing |
J Appl Glycosci (1999)
December 2023
1 Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University.
Some probiotics including lactobacilli, colonize host animal cells by targeting glycosaminoglycans (GAGs), such as heparin, located in the extracellular matrix. Recent studies have shown that several lactic acid bacteria degrade GAGs. Here we show the structure/function relationship of 4-deoxy-L--5-hexosulose-uronate ketol-isomerase (KduI) crucial for metabolism of unsaturated glucuronic acid produced through degradation of GAGs.
View Article and Find Full Text PDFJ Integr Plant Biol
May 2021
State Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing, 210095, China.
A series of nucleotide sugar interconversion enzymes (NSEs) generate the activated sugar donors required for biosynthesis of cell wall matrix polysaccharides and glycoproteins. UDP-glucose 4-epimerases (UGEs) are NSEs that function in the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal). The roles of UDP-glucose 4-epimerases in monocots remain unclear due to redundancy in the pathways.
View Article and Find Full Text PDFJ Ind Microbiol Biotechnol
December 2020
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
A microbial production platform has been developed in Escherichia coli to synthesize D-glyceric acid from D-galacturonate. The expression of uronate dehydrogenase (udh) from Pseudomonas syringae and galactarolactone isomerase (gli) from Agrobacterium fabrum, along with the inactivation of garK, encoding for glycerate kinase, enables D-glyceric acid accumulation by utilizing the endogenous expression of galactarate dehydratase (garD), 5-keto-4-deoxy-D-glucarate aldolase (garL), and 2-hydroxy-3-oxopropionate reductase (garR). Optimization of carbon flux through the elimination of competing metabolic pathways led to the development of a ΔgarKΔhyiΔglxKΔuxaC mutant strain that produced 4.
View Article and Find Full Text PDFJ Biol Chem
August 2020
Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy
UDP-glucuronic acid is converted to UDP-galacturonic acid en route to a variety of sugar-containing metabolites. This reaction is performed by a NAD-dependent epimerase belonging to the short-chain dehydrogenase/reductase family. We present several high-resolution crystal structures of the UDP-glucuronic acid epimerase from The geometry of the substrate-NAD interactions is finely arranged to promote hydride transfer.
View Article and Find Full Text PDFFEBS J
February 2021
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Austria.
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.
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