Determinants of the Nucleotide Specificity in the Carbohydrate Epimerase Family 1

Abstract

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.

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

“…Therefore, we refined the alignment by selecting only crystal structures of enzymes active on NDP-sugars (173 structures in the PDB), giving rise to 28 subfamilies (Table 1). In light of our long-standing interest in carbohydrate epimerases (Beerens et al, 2017;Gevaert et al, 2019;Van Overtveldt et al, 2015) and more specifically in epimerases active on the second asymmetric carbon (Rapp et al, 2020;Van Overtveldt et al, 2020), which still hold unraveled potential, we targeted the CDP-paratose 2-epimerase from Salmonella J o u r n a l P r e -p r o o f typhi, stTyvE (PDB: 1ORR) as template for the new alignment, leading to a core composed of every position of the stTyvE protein sequence (Fig. 2).…”

“…Although it is still not known why, the choice of NDP moiety is conserved for each stereospecificity, raising questions about different mechanisms. However, it has recently been shown that GalE or TyvE from Thermus thermophilus (ttGalE) and Thermodesulfatator atlanticus (taTyvE) respectively, could still perform the epimerization reaction on different NDP-moieties, albeit with different degree of affinity (Rapp et al, 2020;Van Overtveldt et al, 2020). Regarding C3,5-epimerisation, the C4 is oxidized but other extra catalytic residues are needed such as C145 and K217 in Arabidopsis thaliana GM35E.…”

Biocatalytic Approaches to Building Blocks for Enzymatic and Chemical Glycan Synthesis

“…Furthermore, RmlC from S. syringae accepts dTDP-4-keto-6-deoxy- d -Glc and is a key NDP-sugar for the biosynthetic pathway toward dTDP- l -Rha. , Recent work to explore the structure–function relationships in NDP-sugar-active short-chain dehydrogenase/reductase (SDR) enzymes has allowed the identification of patterns of conservation and critical residues, which may serve as a guide for the rational design of SDR enzymes to further expand our capability to access both natural and unnatural NDP-sugars. , …”

Hydride Transfer Mechanism of Enzymatic Sugar Nucleotide C2 Epimerization Probed with a Loose-Fit CDP-Glucose Substrate

“…The thermophilic C2 epimerase from Thermodesulfatator atlanticus ( Ta CPa2E) , was investigated here in combination with a slow CDP-glucose (CDP-Glc) substrate (Scheme a). From its sequence, Ta CPa2E belongs to the group of CDP-paratose/CDP-tyvelose epimerases ,, and is a member of the short-chain dehydrogenase/reductase (SDR) protein superfamily. − The enzyme is a homodimer and each subunit contains tightly bound NAD + .…”

“…KIEs and their temperature dependence can serve as probes of protein motions that affect the C−H bond activation in enzymatic hydride transfer reactions. 13,27,33,34,70,71 The thermophilic C2 epimerase from Thermodesulfatator atlanticus (TaCPa2E) 72,73 was investigated here in combination with a slow CDP-glucose (CDP-Glc) substrate (Scheme 1a). From its sequence, TaCPa2E belongs to the group of CDP-paratose/CDP-tyvelose epimerases 62,66,74 and is a member of the short-chain dehydrogenase/reductase (SDR) protein superfamily.…”