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.

GDP-Mannose 3,5-Epimerase: A View on Structure, Mechanism, and Industrial Potential.

GDP-mannose 3,5-epimerase (GM35E, GME) belongs to the short-chain dehydrogenase/reductase (SDR) protein superfamily and catalyses the conversion of GDP-d-mannose towards GDP-l-galactose. Although the overall reaction seems relatively simple (a double epimerization), the enzyme needs to orchestrate a complex set of chemical reactions, with no less than 6 catalysis steps (oxidation, 2x deprotonation, 2x protonation and reduction), to perform the double epimerization of GDP-mannose to GDP-l-galactose. The enzyme is involved in the biosynthesis of vitamin C in plants and lipopolysaccharide synthesis in bacteria. In this review, we provide a clear overview of these interesting epimerases, including the latest findings such as the recently characterized bacterial and thermostable GM35E representative and its mechanism revision but also focus on their industrial potential in rare sugar synthesis and glycorandomization.

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.