Isotope Probing of the UDP-Apiose/UDP-Xylose Synthase Reaction: Evidence of a Mechanism via a Coupled Oxidation and Aldol Cleavage.

The C-branched sugar d-apiose (Api) is essential for plant cell-wall development. An enzyme-catalyzed decarboxylation/pyranoside ring-contraction reaction leads from UDP-α-d-glucuronic acid (UDP-GlcA) to the Api precursor UDP-α-d-apiose (UDP-Api). We examined the mechanism of UDP-Api/UDP-α-d-xylose synthase (UAXS) with site-selectively 2 H-labeled and deoxygenated substrates. The analogue UDP-2-deoxy-GlcA, which prevents C-2/C-3 aldol cleavage as the plausible initiating step of pyranoside-to-furanoside conversion, did not give the corresponding Api product. Kinetic isotope effects (KIEs) support an UAXS mechanism in which substrate oxidation by enzyme-NAD+ and retro-aldol sugar ring-opening occur coupled in a single rate-limiting step leading to decarboxylation. Rearrangement and ring-contracting aldol addition in an open-chain intermediate then give the UDP-Api aldehyde, which is intercepted via reduction by enzyme-NADH.

Enzymatic Redox Cascade for One-Pot Synthesis of Uridine 5'-Diphosphate Xylose from Uridine 5'-Diphosphate Glucose.

Synthetic ways towards uridine 5'-diphosphate (UDP)-xylose are scarce and not well established, although this compound plays an important role in the glycobiology of various organisms and cell types. We show here how UDP-glucose 6-dehydrogenase (hUGDH) and UDP-xylose synthase 1 (hUXS) from Homo sapiens can be used for the efficient production of pure UDP-α-xylose from UDP-glucose. In a mimic of the natural biosynthetic route, UDP-glucose is converted to UDP-glucuronic acid by hUGDH, followed by subsequent formation of UDP-xylose by hUXS. The nicotinamide adenine dinucleotide (NAD+) required in the hUGDH reaction is continuously regenerated in a three-step chemo-enzymatic cascade. In the first step, reduced NAD+ (NADH) is recycled by xylose reductase from Candida tenuis via reduction of 9,10-phenanthrenequinone (PQ). Radical chemical re-oxidation of this mediator in the second step reduces molecular oxygen to hydrogen peroxide (H2O2) that is cleaved by bovine liver catalase in the last step. A comprehensive analysis of the coupled chemo-enzymatic reactions revealed pronounced inhibition of hUGDH by NADH and UDP-xylose as well as an adequate oxygen supply for PQ re-oxidation as major bottlenecks of effective performance of the overall multi-step reaction system. Net oxidation of UDP-glucose to UDP-xylose by hydrogen peroxide (H2O2) could thus be achieved when using an in situ oxygen supply through periodic external feed of H2O2 during the reaction. Engineering of the interrelated reaction parameters finally enabled production of 19.5 mM (10.5 g l-1) UDP-α-xylose. After two-step chromatographic purification the compound was obtained in high purity (>98%) and good overall yield (46%). The results provide a strong case for application of multi-step redox cascades in the synthesis of nucleotide sugar products.

Structure and mechanism of human UDP-xylose synthase: evidence for a promoting role of sugar ring distortion in a three-step catalytic conversion of UDP-glucuronic acid.

UDP-xylose synthase (UXS) catalyzes decarboxylation of UDP-D-glucuronic acid to UDP-xylose. In mammals, UDP-xylose serves to initiate glycosaminoglycan synthesis on the protein core of extracellular matrix proteoglycans. Lack of UXS activity leads to a defective extracellular matrix, resulting in strong interference with cell signaling pathways. We present comprehensive structural and mechanistic characterization of the human form of UXS. The 1.26-Å crystal structure of the enzyme bound with NAD(+) and UDP reveals a homodimeric short-chain dehydrogenase/reductase (SDR), belonging to the NDP-sugar epimerases/dehydratases subclass. We show that enzymatic reaction proceeds in three chemical steps via UDP-4-keto-D-glucuronic acid and UDP-4-keto-pentose intermediates. Molecular dynamics simulations reveal that the D-glucuronyl ring accommodated by UXS features a marked (4)C(1) chair to B(O,3) boat distortion that facilitates catalysis in two different ways. It promotes oxidation at C(4) (step 1) by aligning the enzymatic base Tyr(147) with the reactive substrate hydroxyl and it brings the carboxylate group at C(5) into an almost fully axial position, ideal for decarboxylation of UDP-4-keto-D-glucuronic acid in the second chemical step. The protonated side chain of Tyr(147) stabilizes the enolate of decarboxylated C(4) keto species ((2)H(1) half-chair) that is then protonated from the Si face at C(5), involving water coordinated by Glu(120). Arg(277), which is positioned by a salt-link interaction with Glu(120), closes up the catalytic site and prevents release of the UDP-4-keto-pentose and NADH intermediates. Hydrogenation of the C(4) keto group by NADH, assisted by Tyr(147) as catalytic proton donor, yields UDP-xylose adopting the relaxed (4)C(1) chair conformation (step 3).

Scale-up and intensification of (S)-1-(2-chlorophenyl)ethanol bioproduction: economic evaluation of whole cell-catalyzed reduction of o-chloroacetophenone.

Escherichia coli cells co-expressing genes coding for Candida tenuis xylose reductase and Candida boidinii formate dehydrogenase were used for the bioreduction of o-chloroacetophenone with in situ coenzyme recycling. The product, (S)-1-(2-chlorophenyl)ethanol, is a key chiral intermediate in the synthesis of polo-like kinase 1 inhibitors, a new class of chemotherapeutic drugs. Production of the alcohol in multi-gram scale requires intensification and scale-up of the biocatalyst production, biotransformation, and downstream processing. Cell cultivation in a 6.9-L bioreactor led to a more than tenfold increase in cell concentration compared to shaken flask cultivation. The resultant cells were used in conversions of 300 mM substrate to (S)-1-(2-chlorophenyl)ethanol (e.e. >99.9%) in high yield (96%). Results obtained in a reaction volume of 500 mL were identical to biotransformations carried out in 1 mL (analytical) and 15 mL (preparative) scale. Optimization of product isolation based on hexane extraction yielded 86% isolated product. Biotransformation and extraction were accomplished in a stirred tank reactor equipped with pH and temperature control. The developed process lowered production costs by 80% and enabled (S)-1-(2-chlorophenyl)ethanol production within previously defined economic boundaries. A simple and efficient way to synthesize (S)-1-(2-chlorophenyl)ethanol in an isolated amount of 20 g product per reaction batch was demonstrated.

Binding pattern of intermediate UDP-4-keto-xylose to human UDP-xylose synthase: Synthesis and STD NMR of model keto-saccharides.

Human UDP-xylose synthase (hUXS1) exclusively converts UDP-glucuronic acid to UDP-xylose via intermediate UDP-4-keto-xylose (UDP-Xyl-4O). Synthesis of model compounds like methyl-4-keto-xylose (Me-Xyl-4O) is reported to investigate the binding pattern thereof to hUXS1. Hence, selective oxidation of the desired hydroxyl function required employment of protecting group chemistry. Solution behavior of synthesized keto-saccharides was studied without enzyme via 1H and 13C NMR spectroscopy with respect to existent forms in deuterated potassium phosphate buffer. Keto-enol tautomerism was observed for all investigated keto-saccharides, while gem-diol hydrate forms were only observed for 4-keto-xylose derivatives. Saturation transfer difference (STD) NMR was used to study binding of synthesized keto-gylcosides to wild type hUXS1. Resulting epitope maps were correlated to earlier published molecular modeling studies of UDP-Xyl-4O. STD NMR results of Me-Xyl-4O are in good agreement with simulations of the intermediate UDP-Xyl-4O indicating a strong interaction of proton H3 with the enzyme, potentially caused by active site residue Ala79. In contrast, pyranoside binding pattern studies of methyl uronic acids showed some differences compared to previously published STD NMR results of UDP-glycosides. In general, obtained results can contribute to a better understanding in binding of UDP-glycosides to other UXS enzyme family members, which have high structural similarities in the active site.

Probing of the reaction pathway of human UDP-xylose synthase with site-directed mutagenesis.

Uridine 5'-diphosphate (UDP)-xylose (UDP-Xyl) synthase (UXS) catalyzes the oxidative decarboxylation of UDP-glucuronic acid (UDP-GlcUA) to UDP-Xyl. The closely related UDP-glucuronic acid 4-epimerase (UGAE) interconverts UDP-GlcUA and UDP-galacturonic acid (UDP-GalUA) in a highly similar manner via the intermediate UDP-xylo-hexopyranos-4-uluronic acid (UDP-4-keto-GlcUA). Unlike UXS, however, UGAE prevents the decarboxylation. Human UXS (hUXS) and UGAE from Arabidopsis thaliana exhibit high structural similarity in the active site, but two catalytically important residues in hUXS (Glu(120) and Arg(277)) are replaced by Ser and Thr in the UGAE group. Additionally, Asn(176), which participates in substrate binding, is changed to Thr. We therefore analyzed single, double and triple mutants of hUXS carrying these substitutions to evaluate their significance for product specificity. All mutants showed considerably lower activities than wild-type hUXS (>1000-fold reduction). NMR spectroscopic analysis of the reaction products showed that UDP-ß-L-threo-pentopyranos-4-ulose (UDP-4-keto-Xyl), UDP-Xyl or both, but no UDP-GalUA or UDP-4-keto-GlcUA were formed. Correlation of product characteristics, such as deuterium incorporation, with the amino acid replacements gave insights into structure-function relationships in UXS, suggesting that interaction between active site and overall enzyme structure rather than distinct conserved residues are decisive for product formation.

Catalytic mechanism of human UDP-glucose 6-dehydrogenase: in situ proton NMR studies reveal that the C-5 hydrogen of UDP-glucose is not exchanged with bulk water during the enzymatic reaction.

Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes the biosynthetic oxidation of UDP-glucose into UDP-glucuronic acid. The catalytic reaction proceeds in two NAD(+)-dependent steps via covalent thiohemiacetal and thioester enzyme intermediates. Formation of the thiohemiacetal adduct occurs through attack of Cys(276) on C-6 of the UDP-gluco-hexodialdose produced in the first oxidation step. Because previous studies of the related enzyme from bovine liver had suggested loss of the C-5 hydrogen from UDP-gluco-hexodialdose due to keto-enol tautomerism, we examined incorporation of solvent deuterium into product(s) of UDP-glucose oxidation by hUGDH. We used wild-type enzyme and a slow-reacting Glu(161)→Gln mutant that accumulates the thioester adduct at steady state. In situ proton NMR measurements showed that UDP-glucuronic acid was the sole detectable product of both enzymatic transformations. The product contained no deuterium at C-5 within the detection limit (≤2%). The results are consistent with the proposed mechanistic idea for hUGDH that incipient UDP-gluco-hexodialdose is immediately trapped by thiohemiacetal adduct formation.