Alexander Gutmann

Leloir Glycosyltransferases and Natural Product Glycosylation: Biocatalytic Synthesis of the C-Glucoside Nothofagin, a Major Antioxidant of Redbush Herbal Tea

Nothofagin is a major antioxidant of redbush herbal tea and represents a class of bioactive flavonoid-like C-glycosidic natural products. We developed an efficient enzymatic synthesis of nothofagin based on a one-pot coupled glycosyltransferase-catalyzed transformation that involves perfectly selective 3′-C-β-d-glucosylation of naturally abundant phloretin and applies sucrose as expedient glucosyl donor. C-Glucosyltransferase from Oryza sativa (rice) was used for phloretin C-glucosylation from uridine 5′-diphosphate (UDP)-glucose, which was supplied continuously in situ through conversion of sucrose and UDP catalyzed by sucrose synthase from Glycine max (soybean). In an evaluation of thermodynamic, kinetic, and stability parameters of the coupled enzymatic reactions, poor water solubility of the phloretin acceptor substrate was revealed as a major bottleneck of conversion efficiency. Using periodic feed of phloretin controlled by reaction progress, nothofagin concentrations (45 mM; 20 g l−1) were obtained that vastly exceed the phloretin solubility limit (5–10 mM). The intermediate UDP-glucose was produced from catalytic amounts of UDP (1.0 mM) and was thus recycled 45 times in the process. Benchmarked against comparable glycosyltransferase-catalyzed transformations (e.g., on quercetin), the synthesis of nothofagin has achieved intensification in glycosidic product formation by up to three orders of magnitude (μM→mM range). It thus makes a strong case for the application of Leloir glycosyltransferases in biocatalytic syntheses of glycosylated natural products as fine chemicals.

Switching between O- and C-Glycosyltransferase through Exchange of Active-Site Motifs†

Many natural products derive their biological activity from the sugars attached to their structure. Their glycosylation therefore often determines efficacy in drug use. Engineering the glycosylation pattern of natural products constitutes a promising way of developing new bioactive molecules with tailored pharmacological properties. Diversification of the glycosylation potentially involves exchange of sugar molecules, but also alterations in type and position of the glycosidic bond(s). Most glycosylated natural products are O-glycosylated; however, N-, C-, and S-glycosides are also known. Chemically, C-glycosides are outstanding in this class because of their pronounced stability to spontaneous and enzyme-catalyzed hydrolysis. C-glycosides have therefore aroused particular interest for medicinal applications where use as isofunctional analogues of the corresponding Oglycosides potentially offers the important advantage of enhanced in vivo half-life. Glycosylations in the biosyntheses of natural products are catalyzed by glycosyltransferases (GTs; EC 2.4). These enzymes use an activated donor substrate, typically a nucleoside diphosphate sugar (e.g. UDP-glucose in Scheme 1), for transferring a glycosyl residue onto the reactive group of an acceptor substrate. GTs show exquisite substrate selectivity and are generally recognized as powerful glycosylation tools for both in vitro and in vivo use. However, GTs naturally capable of forming C-glycosidic bonds (CGTs) appear to be sparse, limiting their availability and scope for synthesis. Therefore, development of useful C-glycosylation catalysts may have to start from existing O-glycosyltransferases (OGTs) using protein engineering. Unfortunately, in contrast to enzymatic O-glycosyl transfer which has been studied in great detail, the mechanistic principles underlying the corresponding C-glycosyl transfer are not well understood, and this presents severe restriction to enzyme design approaches. GT structural features critical for differentiating between Cand O-glycosyl transfer are not known. A significant paper from Bechthold and colleagues recently demonstrated the swapping of glycosidic bond-type specificity from a bacterial aryl-CGT to a structurally and functionally homologous OGT. Through extensive generation of OGT chimeras that harbored distinct sequence elements from the native CGT, a complete OGT-to-CGT switch was eventually obtained. From protein modeling studies, relevant substitutions in engineered CGT were located within activesite loops that were proposed to adopt highly flexible conformations. However, because of the relatively large number of residue substitutions ( 10) required in the native OGT, the molecular interpretation of the specificity change was difficult and a replicable design principle for OGT-to-CGT conversion remained elusive. We report here the implementation of a reciprocal switch in glycosidic bond-type specificity within a homologous pair Scheme 1. Formally proposed mechanisms of enzymatic C-glycosylation adapted to the herein examined glucosylation of phloretin by CGT (in green). C-glycosylation by means of O/C rearrangement involves O-glycosylation, as catalyzed by OGT (orange), in the first step. Activation of the aryl acceptor substrate by a conserved His is essential in both CGT and OGT.

Oxidation of Monolignols by Members of the Berberine Bridge Enzyme Family Suggests a Role in Plant Cell Wall Metabolism

Background: Berberine bridge enzyme-like proteins are a multigene family in plants. Results: Members of the berberine bridge enzyme-like family were identified as monolignol oxidoreductases. Conclusion: Berberine bridge enzyme-like enzymes play a role in monolignol metabolism and lignin formation. Significance: Our results indicate a novel and unexpected role of berberine bridge enzyme-like enzymes in plant biochemistry and physiology. Plant genomes contain a large number of genes encoding for berberine bridge enzyme (BBE)-like enzymes. Despite the widespread occurrence and abundance of this protein family in the plant kingdom, the biochemical function remains largely unexplored. In this study, we have expressed two members of the BBE-like enzyme family from Arabidopsis thaliana in the host organism Komagataella pastoris. The two proteins, termed AtBBE-like 13 and AtBBE-like 15, were purified, and their catalytic properties were determined. In addition, AtBBE-like 15 was crystallized and structurally characterized by x-ray crystallography. Here, we show that the enzymes catalyze the oxidation of aromatic allylic alcohols, such as coumaryl, sinapyl, and coniferyl alcohol, to the corresponding aldehydes and that AtBBE-like 15 adopts the same fold as vanillyl alcohol oxidase as reported previously for berberine bridge enzyme and other FAD-dependent oxidoreductases. Further analysis of the substrate range identified coniferin, the glycosylated storage form of coniferyl alcohol, as a substrate of the enzymes, whereas other glycosylated monolignols were rather poor substrates. A detailed analysis of the motifs present in the active sites of the BBE-like enzymes in A. thaliana suggested that 14 out of 28 members of the family might catalyze similar reactions. Based on these findings, we propose a novel role of BBE-like enzymes in monolignol metabolism that was previously not recognized for this enzyme family.

Sucrose synthase: A unique glycosyltransferase for biocatalytic glycosylation process development

Sucrose synthase (SuSy, EC 2.4.1.13) is a glycosyltransferase (GT) long known from plants and more recently discovered in bacteria. The enzyme catalyzes the reversible transfer of a glucosyl moiety between fructose and a nucleoside diphosphate (NDP) (sucrose+NDP↔NDP-glucose+fructose). The equilibrium for sucrose conversion is pH dependent, and pH values between 5.5 and 7.5 promote NDP-glucose formation. The conversion of a bulk chemical to high-priced NDP-glucose in a one-step reaction provides the key aspect for industrial interest. NDP-sugars are important as such and as key intermediates for glycosylation reactions by highly selective Leloir GTs. SuSy has gained renewed interest as industrially attractive biocatalyst, due to substantial scientific progresses achieved in the last few years. These include biochemical characterization of bacterial SuSys, overproduction of recombinant SuSys, structural information useful for design of tailor-made catalysts, and development of one-pot SuSy-GT cascade reactions for production of several relevant glycosides. These advances could pave the way for the application of Leloir GTs to be used in cost-effective processes. This review provides a framework for application requirements, focusing on catalytic properties, heterologous enzyme production and reaction engineering. The potential of SuSy biocatalysis will be presented based on various biotechnological applications: NDP-sugar synthesis; sucrose analog synthesis; glycoside synthesis by SuSy-GT cascade reactions.

Creating a Water‐Soluble Resveratrol‐Based Antioxidant by Site‐Selective Enzymatic Glucosylation

The phytochemical resveratrol (trans‐3,5,4′‐trihydroxystilbene) has drawn great interest as health‐promoting food ingredient and potential therapeutic agent. However, resveratrol shows vanishingly low water solubility; this limits its uptake and complicates the development of effective therapeutic forms. Glycosylation should be useful to enhance resveratrol solubility, with the caveat that unselective attachment of sugars could destroy the molecules antioxidant activity. UGT71A15 (a uridine 5′‐diphosphate α‐D‐glucose‐dependent glucosyltransferase from apple) was used to synthesize resveratrol 3,5‐β‐D‐diglucoside; this was about 1700‐fold more water‐soluble than the unglucosylated molecule (∼0.18 mM), yet retained most of the antioxidant activity. Resveratrol 3‐β‐D‐glucoside, which is the naturally abundant form of resveratrol, was a practical substrate for perfect site‐selective conversion into the target diglucoside in quantitative yield (g L−1 concentration).

Towards the synthesis of glycosylated dihydrochalcone natural products using glycosyltransferase-catalysed cascade reactions

Regioselective O-β-D-glucosylation of flavonoid core structures is used in plants to create diverse natural products. Their prospective application as functional food and pharmaceutical ingredients makes flavonoid glucosides interesting targets for chemical synthesis, but selective instalment of a glucosyl group requires elaborate synthetic procedures. We report glycosyltransferase-catalysed cascade reactions for single-step highly efficient O-β-D-glucosylation of two major dihydrochalcones (phloretin, davidigenin) and demonstrate their use for the preparation of phlorizin (phloretin 2′-O-β-D-glucoside) and two first-time synthesised natural products, davidioside and confusoside, obtained through selective 2′- and 4′-O-β-D-glucosylation of the dihydroxyphenyl moiety in davidigenin, respectively. Parallel biocatalytic cascades were established by coupling uridine 5′-diphosphate (UDP)-glucose dependent synthetic glucosylations catalysed by herein identified dedicated O-glycosyltransferases (OGTs) to UDP dependent conversion of sucrose by sucrose synthase (SuSy; from soybean). The SuSy reaction served not only to regenerate the UDP-glucose donor substrate for OGT (up to 9 times), but also to overcome thermodynamic restrictions on dihydrochalcone β-D-glucoside formation (up to 20% conversion and yield enhancement). Using conditions optimised for overall coupled enzyme activity, target 2′-O- or 4′-O-β-D-glucoside was obtained in ≥88% yield from reactions consisting of 5 mM dihydrochalcone acceptor, 100 mM sucrose, and 0.5 mM UDP. Davidioside and confusoside were isolated and their proposed chemical structures confirmed by NMR. OGT-SuSy cascade transformations present a green chemistry approach for efficient glucosylation in natural products synthesis.

Screening of recombinant glycosyltransferases reveals the broad acceptor specificity of stevia UGT-76G1

UDP-glycosyltransferases (UGTs) are a promising class of biocatalysts that offer a sustainable alternative for chemical glycosylation of natural products. In this study, we aimed to characterize plant-derived UGTs from the GT-1 family with an emphasis on their acceptor promiscuity and their potential application in glycosylation processes. Recombinant expression in E. coli provided sufficient amounts of enzyme for the in-depth characterization of the salicylic acid UGT from Capsella rubella (UGT-SACr) and the stevia UGT from Stevia rebaudiana (UGT-76G1Sr). The latter was found to have a remarkably broad specificity with activities on a wide diversity of structures, from aliphatic and branched alcohols, over small phenolics to larger flavonoids, terpenoids and even higher glycoside compounds. As an example for its industrial potential, the glycosylation of curcumin was thoroughly evaluated. Under optimized conditions, 96% of curcumin was converted within 24h into the corresponding curcumin β-glycosides. In addition, the reaction was performed in a coupled system with sucrose synthase from Glycine max, to enable the cost-efficient (re)generation of UDP-Glc from sucrose as abundant and renewable resource.

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

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.

Biochemical Characterization and Mechanistic Analysis of the Levoglucosan Kinase from Lipomyces starkeyi

Levoglucosan kinase (LGK) catalyzes the simultaneous hydrolysis and phosphorylation of levoglucosan (1,6‐anhydro‐β‐d‐glucopyranose) in the presence of Mg2+–ATP. For the Lipomyces starkeyi LGK, we show here with real‐time in situ NMR spectroscopy at 10 °C and pH 7.0 that the enzymatic reaction proceeds with inversion of anomeric stereochemistry, resulting in the formation of α‐d‐glucose‐6‐phosphate in a manner reminiscent of an inverting β‐glycoside hydrolase. Kinetic characterization revealed the Mg2+ concentration for optimum activity (20–50 mm), the apparent binding of levoglucosan (Km=180 mm) and ATP (Km=1.0 mm), as well as the inhibition by ADP (Ki=0.45 mm) and d‐glucose‐6‐phosphate (IC50=56 mm). The enzyme was highly specific for levoglucosan and exhibited weak ATPase activity in the absence of substrate. The equilibrium conversion of levoglucosan and ATP lay far on the product side, and no enzymatic back reaction from d‐glucose‐6‐phosphate and ADP was observed under a broad range of conditions. 6‐Phospho‐α‐d‐glucopyranosyl fluoride and 6‐phospho‐1,5‐anhydro‐2‐deoxy‐d‐arabino‐hex‐1‐enitol (6‐phospho‐d‐glucal) were synthesized as probes for the enzymatic mechanism but proved inactive with the enzyme in the presence of ADP. The pyranose ring flip 4C1→1C4 required for 1,6‐anhydro‐product synthesis from d‐glucose‐6‐phosphate probably presents a major thermodynamic restriction to the back reaction of the enzyme.

Sequence determinants of nucleotide binding in Sucrose Synthase : improving the affinity of a bacterial Sucrose Synthase for UDP by introducing plant residues

Sucrose Synthase (SuSy) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate (NDP) into NDP-glucose and fructose. Biochemical characterization of several plant and bacterial SuSys has revealed that the eukaryotic enzymes preferentially use UDP whereas prokaryotic SuSys prefer ADP as acceptor. In this study, SuSy from the bacterium Acidithiobacillus caldus, which has a higher affinity for ADP as reflected by the 25-fold lower Km value compared to UDP, was used as a test case to scrutinize the effect of introducing plant residues at positions in a putative nucleotide binding motif surrounding the nucleobase ring of NDP. All eight single to sextuple mutants had similar activities as the wild-type enzyme but significantly reduced Km values for UDP (up to 60 times). In addition, we recognized that substrate inhibition by UDP is introduced by a methionine at position 637. The affinity for ADP also increased for all but one variant, although the improvement was much smaller compared to UDP. Further characterization of a double mutant also revealed more than 2-fold reduction in Km values for CDP and GDP. This demonstrates the general impact of the motif on nucleotide binding. Furthermore, this research also led to the establishment of a bacterial SuSy variant that is suitable for the recycling of UDP during glycosylation reactions. The latter was successfully demonstrated by combining this variant with a glycosyltransferase in a one-pot reaction for the production of the C-glucoside nothofagin, a health-promoting flavonoid naturally found in rooibos (tea).