UDP-hexose 4-epimerases: a view on structure, mechanism and substrate specificity

Beerens, Koen; Soetaert, Wim; Desmet, Tom

doi:10.1016/j.carres.2015.06.006

Cited by 59 publications

(71 citation statements)

References 71 publications

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“…The results suggested that S121 and Y146 are indispensable for epimerase activity of H3634, whereas in the case of K150R mutant, arginine may be able to partially substitute for lysine leading to a partially active enzyme. These results are in support of the existing reports for homologous proteins, in which Ser and Tyr residues have been observed to be essential for epimerase activity [25][8]. These experiments also support the observation that the source of epimerase activity is indeed the recombinant protein H3634.…”

Section: Resultssupporting

confidence: 92%

“…The first step in the reaction mechanism is abstraction of a hydride. This is followed by the rotation of sugar moiety and transfer of proton back to the sugar giving rise to the epimerised product [8]. Based on a multiple sequence alignment of epimerases with known 3-D structures, residues Ser121, Tyr146 and Lys150 in Rv3634c were identified as putative catalytic residues (Fig 9).…”

Section: Resultsmentioning

confidence: 99%

“…Members of this extended SDR subfamily have a Rossmann fold domain in the N-terminal region and this domain contains the NAD(P) binding motif GxxGxxG. The catalytic site consists of the motif YxxxK in most of the SDR enzymes [8][9]. In general, dehydratases and epimerases have similar sequences and it is not possible to unambiguously annotate them by sequence comparison alone.…”

Section: Introductionmentioning

confidence: 99%

“…aeruginosa Gne) use only UDP-GlcNAc/GalNAc as substrates. Type II (e.g., Homo sapiens Gne) enzymes can utilize both these as substrates [11][8]. …”

Section: Introductionmentioning

confidence: 99%

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Rv3634c from Mycobacterium tuberculosis H37Rv encodes an enzyme with UDP-Gal/Glc and UDP-GalNAc 4-epimerase activities

2017

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A bioinformatics study revealed that Mycobacterium tuberculosis H37Rv (Mtb) contains sequence homologs of Campylobacter jejuni protein glycosylation enzymes. The ORF Rv3634c from Mtb was identified as a sequence homolog of C. jejuni UDP-Gal/GalNAc 4-epimerase. This study reports the cloning of Rv3634c and its expression as an N-terminal His-tagged protein. The recombinant protein was shown to have UDP-Gal/Glc 4-epimerase activity by GOD-POD assay and by reverse phase HPLC. This enzyme was shown to have UDP-GalNAc 4-epimerase activity also. Residues Ser121, Tyr146 and Lys150 were shown by site-directed mutagenesis to be important for enzyme activity. Mutation of Ser121 and Tyr146 to Ala and Phe, respectively, led to complete loss of activity whereas mutation of Lys150 to Arg led to partial loss of activity. There were no gross changes in the secondary structures of any of these three mutants. These results suggest that Ser121 and Tyr146 are essential for epimerase activity of Rv3634c. UDP-Gal/Glc 4-epimerases from other organisms also have a catalytic triad consisting of Ser, Tyr and Lys. The triad carries out proton transfer from nucleotide sugar to NAD+ and back, thus effecting the epimerization of the substrate. Addition of NAD+ to Lys150 significantly abrogates the loss of activity, suggesting that, as in other epimerases, NAD+ is associated with Rv3634c.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…aeruginosa Gne) use only UDP-GlcNAc/GalNAc as substrates. Type II (e.g., Homo sapiens Gne) enzymes can utilize both these as substrates [11][8]. …”

Section: Introductionmentioning

confidence: 99%

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Rv3634c from Mycobacterium tuberculosis H37Rv encodes an enzyme with UDP-Gal/Glc and UDP-GalNAc 4-epimerase activities

2017

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“…The corresponding ugeA gene in A. nidulans was studied previously for its anabolic function in providing the uridylylated galactose monomers for cell wall galactofurans [12]. UDP-hexose 4-epimerases often accept both UDP hexoses and their N-acetylated 2-amino forms as well as UDP-pentoses as their substrate (reviewed by [3]. In A. fumigatus, two structurally related UDP-hexose 4-epimerases were recently shown to be required for the synthesis of the galactosaminogalactan exopolysaccharide content of the fungal cell wall [26].…”

Section: Growth Of Penicillium Chrysogenum On D-galactosementioning

confidence: 99%

D-galactose catabolism inPenicillium chrysogenum: Expression analysis of the structural genes of the Leloir pathway

Jónás

Fekete

Németh

et al. 2016

Acta Biologica Hungarica

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In this study, we analyzed the expression of the structural genes encoding the five enzymes comprising the Leloir pathway of D-galactose catabolism in the industrial cell factory Penicillium chrysogenum on various carbon sources. The genome of P. chrysogenum contains a putative galactokinase gene at the annotated locus Pc13g10140, the product of which shows strong structural similarity to yeast galactokinase that was expressed on lactose and D-galactose only. The expression profile of the galactose-1-phosphate uridylyl transferase gene at annotated locus Pc15g00140 was essentially similar to that of galactokinase. This is in contrast to the results from other fungi such as Aspergillus nidulans, Trichoderma reesei and A. niger, where the ortholog galactokinase and galactose-1-phosphate uridylyl transferase genes were constitutively expressed. As for the UDP-galactose-4-epimerase encoding gene, five candidates were identified. We could not detect Pc16g12790, Pc21g12170 and Pc20g06140 expression on any of the carbon sources tested, while for the other two loci (Pc21g10370 and Pc18g01080) transcripts were clearly observed under all tested conditions. Like the 4-epimerase specified at locus Pc21g10370, the other two structural Leloir pathway genes -UDP-glucose pyrophosphorylase (Pc21g12790) and phosphoglucomutase (Pc18g01390) -were expressed constitutively at high levels as can be expected from their indispensable function in fungal cell wall formation.

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Enzymatische C4‐Epimerisierung von UDP‐Glucuronsäure: präzise gesteuerte Rotation eines transienten 4‐Ketointermediats für eine invertierende Reaktion ohne Decarboxylierung

Borg

Esquivias²,

Coines³

et al. 2022

Angewandte Chemie

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UDP-Glucuronsäure(UDP-GlcA)-4-Epimerase repräsentiert eine wichtige Fragestellung in der Enzymkatalyse: die Balance zwischen konformativer Flexibilität und genauer Positionierung. Das Enzym koordiniert die C4-Oxidation des Substrats durch NAD + mit der Rotation eines leicht decarboxylierbaren β-Ketosäure-Intermediats im aktiven Zentrum zur Ermöglichung der stereoinvertierenden Reduktion der Ketogruppe durch NADH. Wir zeigen hier die nur schwer erfassbare Rotationskoordinate des 4-Ketointermediats. Distorsion des Zuckerrings in eine Boot-Konformation erzeugt torsionale Mobilität in der Bindungstasche des Enzyms. Die Endpunkte der Rotation zeigen den 4-Ketozucker in einer unverformten 4 C 1 -Sesselkonformation. Die äquatorial positionierte Carboxylatgruppe ist ungünstig für die 4-Ketozucker-Decarboxylierung. Varianten der Epimerase zeigen Decarboxylierung, wenn sie die Bindung mit der Carboxylatgruppe im entgegengesetzten Rotationsisomer des Substrats entfernen. R185A/D-Substitutionen wandeln die Epimerase in UDP-Xylose-Synthasen um, welche UDP-GlcA in stereospezifischen, konfigurationserhaltenden Reaktionen decarboxylieren.

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UDP-hexose 4-epimerases: a view on structure, mechanism and substrate specificity

Cited by 59 publications

References 71 publications

Rv3634c from Mycobacterium tuberculosis H37Rv encodes an enzyme with UDP-Gal/Glc and UDP-GalNAc 4-epimerase activities

Rv3634c from Mycobacterium tuberculosis H37Rv encodes an enzyme with UDP-Gal/Glc and UDP-GalNAc 4-epimerase activities

D-galactose catabolism inPenicillium chrysogenum: Expression analysis of the structural genes of the Leloir pathway

Enzymatische C4‐Epimerisierung von UDP‐Glucuronsäure: präzise gesteuerte Rotation eines transienten 4‐Ketointermediats für eine invertierende Reaktion ohne Decarboxylierung

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