AbstractDer C‐verzweigte Zucker d‐Apiose (Api) ist essenziell für die Entwicklung der pflanzlichen Zellwand. Eine enzymkatalysierte Decarboxylierungs‐Pyranosidringkontraktions‐Reaktion führt von UDP‐α‐d‐Glucuronsäure (UDP‐GlcA) zur Api‐Vorstufe UDP‐α‐d‐Apiose (UDP‐Api). Der Mechanismus von UDP‐Api/UDP‐α‐d‐Xylose‐Synthase (UAXS) wurde mittels ortsspezifisch 2H‐markierter sowie desoxygenierter Substrate untersucht. Aus dem Analogon UDP‐2‐Desoxy‐GlcA, das die C‐2/C‐3‐Aldolspaltung als den wahrscheinlichen Schritt zur Einleitung der Pyranosid‐zu‐Furanosid‐Umwandlung verhindert, bildete sich kein entsprechendes Api‐Produkt. Die kinetischen Isotopeneffekte stützen einen UAXS‐Mechanismus, in dem die Substratoxidation durch Enzym‐NAD+ und die Retroaldol‐Zuckerringöffnung gekoppelt in einem einzelnen geschwindigkeitsbestimmenden Schritt auftreten, der zur Decarboxylierung führt. Eine Neuanordnung und ringkontrahierende Aldoladdition in einem offenkettigen Zwischenprodukt ergeben dann UDP‐Api‐Aldehyd, der durch Enzym‐NADH reduziert wird.

Abstract 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 2H‐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.

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.

A novel Saccharomyces cerevisiae whole-cell biocatalyst for xylitol production based on Candida tenuis xylose reductase (CtXR) is presented. Six recombinant strains expressing wild-type CtXR or an NADH-specific mutant were constructed and evaluated regarding effects of expression mode, promoter strength, biocatalyst concentration and medium composition. Intracellular XR activities ranged from 0.09 U mg(Prot)(1) to 1.05 U mg(Prot)(1) but did not correlate with the strains' xylitol productivities, indicating that other factors limited xylose conversion in the high-activity strains. The CtXR mutant decreased the biocatalyst's performance, suggesting use of the NADPH-preferring wild-type enzyme when (semi-)aerobic conditions are applied. In a bioreactor process, the best-performing strain converted 40 g L-1 xylose with an initial productivity of 1.16 g L-1 h(-1) and a xylitol yield of 100%. The obtained results underline the potential of CtXR wild-type for xylose reduction and point out parameters to improve "green" xylitol production. (C) 2015 Elsevier Ltd. All rights reserved.

The human form of UDP-xylose synthase (hUXS1A) is studied with respect to its substrate and co-enzyme binding in binary and ternary complexes using saturation transfer difference (STD) NMR and in situ NMR. Obtained binding pattern results are correlated to the recently solved crystal structure of hUXS1A and docking studies of UDP-GlcUA, providing a better understanding of substrate specificity of this enzyme and may give useful information in mutant designing. In unproductive binary complexes UDP-saccharide aglycone moieties show strong STD effects with the protein. In contrast, pyranoside rings (Glc, GlcUA, Gal) indicate less interaction with the hUXS1A active site, which enables the required ring distortion of the pyranoside ring in UDP-GlcUA. In productive ternary complexes UDP-GlcUA possesses reasonable binding, while produced UDP-Xyl shows smaller STD responses and does not efficiently compete with the substrate for binding at the active site. STD NMR derived binding studies of NAD(+) demonstrate tight interaction between co-factor and hUXS1A. Higher magnetization of NAD(+) in the presence of enzymatic product is observed and suggests increased contact with groups on the protein. Furthermore, binding studies of substrate analogues having the same stereochemistry as the investigated UDP-saccharides and a small aglycone residue indicate a different mode of action, not guided by the anchor groups.

The human form of UDP-xylose synthase (hUXS1A) is studied with respect to its substrate and co-enzyme binding in binary and ternary complexes using saturation transfer difference (STD) NMR and in situ NMR. Obtained binding pattern results are correlated to the recently solved crystal structure of hUXS1A and docking studies of UDP-GlcUA, providing a better understanding of substrate specificity of this enzyme and may give useful information in mutant designing. In unproductive binary complexes UDPsaccharide aglycone moieties show strong STD effects with the protein. In contrast, pyranoside rings (Glc, GlcUA, Gal) indicate less interaction with the hUXS1A active site, which enables the required ring distortion of the pyranoside ring in UDP-GlcUA. In productive ternary complexes UDP-GlcUA possesses reasonable binding, while produced UDP-Xyl shows smaller STD responses and does not efficiently compete with the substrate for binding at the active site. STD NMR derived binding studies of NAD demonstrate tight interaction between co-factor and hUXS1A. Higher magnetization of NAD in the presence of enzymatic product is observed and suggests increased contact with groups on the protein. Furthermore, binding studies of substrate analogues having the same stereochemistry as the investigated UDP-saccharides and a small aglycone residue indicate a different mode of action, not guided by the anchor groups.

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-β-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.

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.

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.

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.

Chemie Ingenieur TechnikVolume 84, Issue 8 p. 1193-1193 VortragFree Access Prozessentwicklung für die Bioreduktion von o-Chloracetophenon: Strategien zur Überwindung von Substrat- und Produkttoxizität Dr. R. Kratzer, Corresponding Author Dr. R. Kratzer regina.kratzer@tugraz.at Technische Universität Graz, Institut für Biotechnologie und Bioprozesstechnik, Petersgasse 12, A-8010 Graz, AustriaTechnische Universität Graz, Institut für Biotechnologie und Bioprozesstechnik, Petersgasse 12, A-8010 Graz, AustriaSearch for more papers by this authorC. Gruber, C. Gruber Technische Universität Graz, Institut für Biotechnologie und Bioprozesstechnik, Petersgasse 12, A-8010 Graz, AustriaSearch for more papers by this authorT. Eixelsberger, T. Eixelsberger Technische Universität Graz, Institut für Biotechnologie und Bioprozesstechnik, Petersgasse 12, A-8010 Graz, AustriaSearch for more papers by this authorProf. B. Nidetzky, Prof. B. Nidetzky Technische Universität Graz, Institut für Biotechnologie und Bioprozesstechnik, Petersgasse 12, A-8010 Graz, AustriaSearch for more papers by this author Dr. R. Kratzer, Corresponding Author Dr. R. Kratzer regina.kratzer@tugraz.at Technische Universität Graz, Institut für Biotechnologie und Bioprozesstechnik, Petersgasse 12, A-8010 Graz, AustriaTechnische Universität Graz, Institut für Biotechnologie und Bioprozesstechnik, Petersgasse 12, A-8010 Graz, AustriaSearch for more papers by this authorC. Gruber, C. Gruber Technische Universität Graz, Institut für Biotechnologie und Bioprozesstechnik, Petersgasse 12, A-8010 Graz, AustriaSearch for more papers by this authorT. Eixelsberger, T. Eixelsberger Technische Universität Graz, Institut für Biotechnologie und Bioprozesstechnik, Petersgasse 12, A-8010 Graz, AustriaSearch for more papers by this authorProf. B. Nidetzky, Prof. B. Nidetzky Technische Universität Graz, Institut für Biotechnologie und Bioprozesstechnik, Petersgasse 12, A-8010 Graz, AustriaSearch for more papers by this author First published: 25 July 2012 https://doi.org/10.1002/cite.201250419AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume84, Issue8Special Issue: ProcessNet-Jahrestagung 2012 und 30. Jahrestagung der BiotechnologenAugust, 2012Pages 1193-1193 RelatedInformation

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).