Molecular evolution and functional divergence of UDP-hexose 4-epimerases

doi:10.1016/j.cbpa.2020.09.007

Current Opinion in Chemical Biology

Volume 61, April 2021, Pages 53-62

https://doi.org/10.1016/j.cbpa.2020.09.007 Get rights and content

Abstract

UDP-glucose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) and/or the interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc) in sugar metabolism. GalEs belong to the short-chain dehydrogenase/reductase superfamily, use a conserved ‘transient keto intermediate’ mechanism and have variable substrate specificity. GalEs have been classified into three groups based on substrate specificity: group 1 prefers UDP-Glc/Gal, group 3 prefers UDP-GlcNAc/GalNAc, and group 2 has comparable activities for both types of the substrates. The phylogenetic relationship and structural basis for the specificities of GalEs revealed possible molecular evolution of UDP-hexose 4-epimerases in various organisms. Based on the recent advances in studies on GalEs and related enzymes, an updated view of their evolutional diversification is presented.

Introduction

UDP-glucose 4-epimerase or UDP-galactose 4-epimerase (GalE, EC 5.1.3.2; the former name is recommended by IUBMB-IUPAC) catalyzes epimerization between UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal). The abbreviated name GalE is also collectively used for epimerases that catalyze oxidoreductive interconversion of UDP-linked gluco- and galacto-hexoses (UDP-hexose 4-epimerases) [1]. Studies of GalE were mainly motivated to understand the molecular basis of galactose metabolism (Leloir pathway), whose genetic disorder can result in a severe human disease called galactosemia [2,3]. Extensive structural studies were performed in the 1990s using GalE from Escherichia coli (EcGalE) [4, 5, 6], and the mechanistic features were studied in detail. In the early 2000s, the structures of GalE from human (HsGalE) [7, 8, 9, 10] and African sleeping sickness parasite (Trypanosoma brucei; TbGalE) [11,12] were also reported. An interesting feature of these GalEs is their substrate specificities. HsGalE is a promiscuous enzyme that can catalyze interconvertion between UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc) and between UDP-Glc and UDP-Gal. In contrast, EcGalE and TbGalE are solely active on UDP-Glc/Gal and have no activity on the N-acetylated substrates. In 2004, the crystal structure of a genuine UDP-GlcNAc 4-epimerase (with very weak activity on UDP-Glc/Gal) from Pseudomonas aeruginosa (PaWbpP) was reported [13]. PaWbpP is involved in the biosynthesis of the O-antigen of lipopolysaccharide, which is a major virulence factor of the pathogenic bacteria. Ishiyama et al. [13] established a classification system of the substrate specificity of GalEs: group 1 enzymes (e.g. EcGalE and TbGalE) prefer UDP-Glc/Gal; group 2 enzymes (e.g. HsGalE) do not have a preference for either UDP-Glc/Gal or UDP-GlcNAc/GalNAc; and group 3 enzymes (e.g. PaWbpP) prefer UDP-GlcNAc/GalNAc. A phylogenetic analysis on GalEs known at that time indicated that they form three major clades [13]. PaWbpP is placed in a divergent clade of group 3, and the two group 1 enzymes are separated in two different clades. TbGalE (group 1a) represents a distinct clade, whereas EcGalE (group 1b) is placed in the same clade with HsGalE (group 2). Recently, a structural analysis of another group 2 enzyme from a human gut symbiont, Bifidobacterium longum, (BlGalE) was reported [14∗]. In this review, an updated overview of the molecular evolution and structural basis for the substrate specificity is presented based on recent advances in structural and mechanistic studies of GalEs and related enzymes from various organisms.

Section snippets

The phylogenetic overview of GalEs and related enzymes

To explore a possible evolutionary history of GalEs and related enzymes, a phylogenetic tree was constructed using the maximum-likelihood method (Figure 1) [15]. In addition to the enzymes and hypothetical proteins that appeared in the original phylogenetic tree presented in 2004 [13], other enzymes were included based on the currently available information. Threonine dehydrogenases, UDP-GlcNAc dehydratase (Bacillus thuringiensis Pen) [16∗], and Cj1427 from Campylobacter jejuni [17] were

Structural overview and catalytic mechanism of GalEs

GalEs and related enzymes share the same protein fold (the SDR fold; Figure 2a). The N-terminal Rossmann fold domain tightly binds the NAD⁺ cofactor as a prosthetic group, and the C-terminal domain is mainly responsible for binding of the UDP-sugar substrate. In the classification system of carbohydrate epimerases, NAD⁺-dependent UDP-sugar epimerases having the SDR fold are classified in the CEP1 family [34]. The CEP1 family enzymes use a conserved mechanism called a ‘transient keto

Clade B: structural basis for the specificity differences of GalEs from gut microbes and human

Figure 3a shows the sugar-binding pocket of EcGalE in the standard (left) and the rotated (right) conformations. GalEs in clade B preferentially bind the UDP-sugar substrates in the standard conformation in crystal structures. In EcGalE, the rotated conformation was observed only when a double mutant at the binding site (S124A/Y149F) was used [6]. Figure 3b shows the superimposition of HsGalE and BlGalE (group 2b enzymes) complexed with UDP-Glc (left) and UDP-GlcNAc (right). The substrate

Group 1a and 2a enzymes in clade A

TbGalE is the representative member of group 1a and has narrow C5 and C2 pockets with bulky Leu342 and His221 residues, respectively (Figure 4a). UDP-4-deoxy-4-fluoro-galactose is bound in a rotated conformation to TbGalE [12]. Substrate-free structures of GalEs from Bacillus anthracis (BAS5114, PDB: 2C20) and Brucella abortus (PDB: 4TWR) are also available in the database. These enzymes from animal pathogens also have bulky Leu and His at the C5 and C2 pockets, respectively, and BAS5114 was

Group 3c enzymes in clade C

PaWbpP is the representative member of group 3c enzymes in clade C, and the crystal structure complexed with UDP-GalNAc in a rotated conformation was reported (Figure 4d) [13]. The genuine specificity of PaWbpP to UDP-GlcNAc/GalNAc was explained by ‘orbital steering’ that is supported by the stabilization of the N-acetyl group of the substrate with ordered solvent molecules in the pocket. In this explanation, the UDP-Glc/Gal substrates may be less restrained in the active site and, thus, less

Studies on GalEs from thermophilic bacteria and archaea reorganized the phylogenetic grouping of the substrate specificity (clades D and E)

Group 2 GalEs from thermophilic bacteria (Thermus, Marinithermus, and Thermotoga species) form the clade D (Figure 1, group 2d). A biochemical study suggested that GalE from Thermus thermophilis (TTHA0591) has a group 2 specificity [49]. TmGalE exhibited comparable activities toward UDP-Glc, UDP-Gal, UDP-GlcNAc, and UDP-GalNAc [21]. GalE from Marinithermus hydrothermalis also displays group 2 activity, but shows slight preference for UDP-Glc/Gal over UDP-GlcNAc/GalNAc [50]. The crystal

Conclusion

In this review, phylogenetic and structural aspects of GalEs were revisited to present a current overview, which is derived from the original concept established in 2004 [13]. A recent study of the enzyme from a symbiotic gut microbe (B. longum) illustrated its intimate structural similarity with the human enzyme. The detailed structural basis for the genuine specificity toward UDP-GlcNAc/GalNAc enzymes was revealed by recent studies on the group 3c enzymes. Studies on enzymes from thermophilic

Declaration of Competing Interest

The author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

The author thanks Drs Young-Woo Nam, Mamoru Nishimoto, Takatoshi Arakawa, and Motomitsu Kitaoka. The study on BlGalE in collaboration with them inspired the concept of this review article. The author also thanks Dr. Chihaya Yamada for her advice on the phylogenetic analysis.

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Molecular evolution and functional divergence of UDP-hexose 4-epimerases

Abstract

Introduction

Section snippets

The phylogenetic overview of GalEs and related enzymes

Structural overview and catalytic mechanism of GalEs

Clade B: structural basis for the specificity differences of GalEs from gut microbes and human

Group 1a and 2a enzymes in clade A

Group 3c enzymes in clade C

Studies on GalEs from thermophilic bacteria and archaea reorganized the phylogenetic grouping of the substrate specificity (clades D and E)

Conclusion

Declaration of Competing Interest

Acknowledgement

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Angew Chem Int Ed

Nucleic Acids Res

Mol Biol Evol

Biochemistry

Carbohydr Res

Nucleic Acids Res

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Biochem Biophys Res Commun

The Leloir pathway: a mechanistic imperative for three enzymes to change the stereochemical configuration of a single carbon in galactose

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Molecular structure of the NADH/UDP-glucose abortive complex of UDP-galactose 4-epimerase from Escherichia coli: implications for the catalytic mechanism

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Molecular structures of the S124A, S124T, and S124V site-directed mutants of UDP-galactose 4-epimerase from Escherichia coli

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Dramatic differences in the binding of UDP-galactose and UDP-glucose to UDP-galactose 4-epimerase from Escherichia coli

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Crystallographic evidence for Tyr 157 functioning as the active site base in human UDP-galactose 4-epimerase

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Human UDP-galactose 4-epimerase

J Biol Chem