Structure-function relationships in NDP-sugar active SDR enzymes: Fingerprints for functional annotation and enzyme engineering.

Short-chain Dehydrogenase/Reductase enzymes that are active on nucleotide sugars (abbreviated as NS-SDR) are of paramount importance in the biosynthesis of rare sugars and glycosides. Some family members have already been extensively characterized due to their direct implication in metabolic disorders or in the biosynthesis of virulence factors. In this review, we combine the knowledge gathered from studies that typically focused only on one NS-SDR activity with an in-depth analysis and overview of all of the different NS-SDR families (169,076 enzyme sequences). Through this structure-based multiple sequence alignment of NS-SDRs retrieved from public databases, we could identify clear patterns in conservation and correlation of crucial residues. Supported by this analysis, we suggest updating and extending the UDP-galactose 4-epimerase "hexagonal box model" to an "heptagonal box model" for all NS-SDR enzymes. This specificity model consists of seven conserved regions surrounding the NDP-sugar substrate that serve as fingerprint for each specificity. The specificity fingerprints highlighted in this review will be beneficial for functional annotation of the large group of NS-SDR enzymes and form a guide for future enzyme engineering efforts focused on the biosynthesis of rare and specialty carbohydrates.

Expanding the Enzyme Repertoire for Sugar Nucleotide Epimerization: The CDP-Tyvelose 2-Epimerase from Thermodesulfatator atlanticus for Glucose/Mannose Interconversion.

Epimerization of sugar nucleotides is central to the structural diversification of monosaccharide building blocks for cellular biosynthesis. Epimerase applicability to carbohydrate synthesis can be limited, however, by the high degree of substrate specificity exhibited by most sugar nucleotide epimerases. Here, we discovered a promiscuous type of CDP-tyvelose 2-epimerase (TyvE)-like enzyme that promotes C2-epimerization in all nucleotide (CDP, UDP, GDP, ADP, TDP)-activated forms of d-glucose. This new epimerase, originating from Thermodesulfatator atlanticus, is a functional homodimer that contains one tightly bound NAD+/subunit and shows optimum activity at 70°C and pH 9.5. The enzyme exhibits a k cat with CDP-dglucose of ∼1.0 min-1 (pH 7.5, 60°C). To characterize the epimerase kinetically and probe its substrate specificity, we developed chemo-enzymatic syntheses for CDP-dmannose, CDP-6-deoxy-dglucose, CDP-3-deoxy-dglucose and CDP-6-deoxy-dxylo-hexopyranos-4-ulose. Attempts to obtain CDP-dparatose and CDP-dtyvelose were not successful. Using high-resolution carbohydrate analytics and in situ NMR to monitor the enzymatic conversions (60°C, pH 7.5), we show that the CDP-dmannose/CDP-dglucose ratio at equilibrium is 0.67 (± 0.1), determined from the kinetic Haldane relationship and directly from the reaction. We further show that deoxygenation at sugar C6 enhances the enzyme activity 5-fold compared to CDP-dglucose whereas deoxygenation at C3 renders the substrate inactive. Phylogenetic analysis places the T. atlanticus epimerase into a distinct subgroup within the sugar nucleotide epimerase family of SDR (short-chain dehydrogenases/reductases), for which the current study now provides the functional context. Collectively, our results expand an emerging toolbox of epimerase-catalyzed reactions for sugar nucleotide synthesis.IMPORTANCE Epimerases of the sugar nucleotide-modifying class of enzymes have attracted considerable interest in carbohydrate (bio)chemistry, for the mechanistic challenges and the opportunities for synthesis involved in the reactions catalyzed. Discovery of new epimerases with expanded scope of sugar nucleotide substrates used is important to promote the mechanistic inquiry and can facilitate the development of new enzyme applications. Here, a CDP-tyvelose 2-epimerase-like enzyme from Thermodesulfatator atlanticus is shown to catalyze sugar C2 epimerization in CDP-glucose and other nucleotide-activated forms of dglucose. The reactions are new to nature in the context of enzymatic sugar nucleotide modification. The current study explores the substrate scope of the discovered C2-epimerase and, based on modeling, suggests structure-function relationships that may be important for specificity and catalysis.

GDP-altrose as novel product of GDP-mannose 3,5-epimerase: Revisiting its reaction mechanism.

GDP-mannose 3,5-epimerase (GM35E) catalyzes the double epimerization of GDP-mannose to yield GDP-l-galactose. GDP-l-gulose (C5-epimer) has previously been detected as a byproduct of this reaction, indicating that C3,5-epimerization occurs through an initial epimerization at C5. Given these products, GM35E constitutes a valuable bridge between d- and l-hexoses. In order to fully exploit this potential, the enzyme might be subjected to specificity engineering for which profound mechanistic insights are beneficial. Accordingly, this study further elucidated GM35E's reaction mechanism. For the first time, the production of the C3-epimer GDP-altrose was demonstrated, resulting in an adjustment of the acknowledged reaction mechanism. As GM35E converts GDP-mannose to GDP-l-gulose, GDP-altrose and GDP-l-galactose in a 72:4:4:20 ratio, this indicates that the enzyme does not discriminate between the C3 and C5 position as initial epimerization site. This was also confirmed by a structural investigation. Based on a mutational analysis of the active site, residues S115 and R281 were attributed a stabilizing function, which is believed to support the reactivation process of the catalytic residues. This paper eventually reflected on some engineering strategies that aim to change the enzyme towards a single specificity.

Determinants of the Nucleotide Specificity in the Carbohydrate Epimerase Family 1.

In recent years, carbohydrate epimerases have attracted increasing attention as promising biocatalysts for the production of specialty sugars and derivatives. The vast majority of these enzymes are active on nucleotide-activated sugars, rather than on their free counterparts. Although such epimerases are known to have a clear preference for a particular nucleotide (UDP, GDP, CDP, or ADP), very little is known about the determinants of the respective specificities. In this work, sequence motifs are identified that correlate with the different nucleotide specificities in one of the main epimerase superfamilies, carbohydrate epimerase 1 (CEP1). To confirm their relevance, GDP- and CDP-specific residues are introduced into the UDP-glucose 4-epimerase from Thermus thermophilus, resulting in a 3-fold and 13-fold reduction in KM for GDP-Glc and CDP-Glc, respectively. Moreover, several variants are severely crippled in UDP-Glc activity, which further underlines the crucial role of the identified positions. Hence, the analysis should prove to be valuable for the further exploration and application of epimerases involved in carbohydrate synthesis.

Characterization of the First Bacterial and Thermostable GDP-Mannose 3,5-Epimerase.

GDP-mannose 3,5-epimerase (GM35E) catalyzes the conversion of GDP-mannose towards GDP-l-galactose and GDP-l-gulose. Although this reaction represents one of the few enzymatic routes towards the production of l-sugars and derivatives, it has not yet been exploited for that purpose. One of the reasons is that so far only GM35Es from plants have been characterized, yielding biocatalysts that are relatively unstable and difficult to express heterologously. Through the mining of sequence databases, we succeeded in identifying a promising bacterial homologue. The gene from the thermophilic organism Methylacidiphilum fumariolicum was codon optimized for expression in Escherichia coli, resulting in the production of 40 mg/L of recombinant protein. The enzyme was found to act as a self-sufficient GM35E, performing three chemical reactions in the same active site. Furthermore, the biocatalyst was highly stable at temperatures up to 55 °C, making it well suited for the synthesis of new carbohydrate products with application in the pharma industry.

Converting Galactose into the Rare Sugar Talose with Cellobiose 2-Epimerase as Biocatalyst.

Cellobiose 2-epimerase from Rhodothermus marinus (RmCE) reversibly converts a glucose residue to a mannose residue at the reducing end of ß-1,4-linked oligosaccharides. In this study, the monosaccharide specificity of RmCE has been mapped and the synthesis of d-talose from d-galactose was discovered, a reaction not yet known to occur in nature. Moreover, the conversion is industrially relevant, as talose and its derivatives have been reported to possess important antimicrobial and anti-inflammatory properties. As the enzyme also catalyzes the keto-aldo isomerization of galactose to tagatose as a minor side reaction, the purity of talose was found to decrease over time. After process optimization, 23 g/L of talose could be obtained with a product purity of 86% and a yield of 8.5% (starting from 4 g (24 mmol) of galactose). However, higher purities and concentrations can be reached by decreasing and increasing the reaction time, respectively. In addition, two engineering attempts have also been performed. First, a mutant library of RmCE was created to try and increase the activity on monosaccharide substrates. Next, two residues from RmCE were introduced in the cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) (S99M/Q371F), increasing the kcat twofold.

A structural classification of carbohydrate epimerases: From mechanistic insights to practical applications.

In recent years, carbohydrate epimerases have attracted a lot of attention as efficient biocatalysts that can convert abundant sugars (e.g.d-fructose) directly into rare counterparts (e.g.d-psicose). Despite increased research activities, no review about these enzymes has been published in more than a decade, meaning that their full potential is hard to appreciate. Here, we present an overview of all known carbohydrate epimerases based on a classification in structural families, which links every substrate specificity to a well-defined reaction mechanism. The mechanism can even be predicted for enzymes that have not yet been characterized or that lack structural information. In this review, the different families are discussed in detail, both structurally and mechanistically, with special reference to recent examples in the literature. Furthermore, the value of understanding the reaction mechanism will be illustrated by making the link to possible application and engineering targets.