title: Characterization and mutational analysis of the UDP-Glc(NAc) 4-epimerase from Marinithermus hydrothermalis.
abstract: UDP-hexose 4-epimerases are important enzymes that play key roles in various biological pathways, including lipopolysaccharide biosynthesis, galactose metabolism through the Leloir pathway, and biofilm formation. Unfortunately, the determinants of their substrate specificity are not yet fully understood. They can be classified into three groups, with groups 1 and 3 preferring non-acetylated and acetylated UDP-hexoses, respectively, whereas members of group 2 are equally active on both types of substrates. In this study, the UDP-Glc(NAc) 4-epimerase from Marinithermus hydrothermalis (mGalE) was functionally expressed in Escherichia coli and thoroughly characterized. The enzyme was found to be thermostable, displaying its highest activity at 70 °C and having a half-life of 23 min at 60 °C. Activity could be detected on both acetylated and non-acetylated UDP-hexoses, meaning that this epimerase belongs to group 2. This observation correlates well with the identity of the so-called "gatekeeper" residue (Ser279), which has previously been suggested to influence substrate specificity (Schulz et al., J Biol Chem 279:32796-32803, 2004). Furthermore, substituting this serine to a tyrosine brings about a significant preference for non-acetylated sugars, thereby demonstrating that a single residue can determine substrate specificity among type 1 and type 2 epimerases. In addition, two consecutive glycine residues (Gly118 and Gly119) were identified as a unique feature of GalE enzymes from Thermus species, and their importance for activity as well as affinity was confirmed by mutagenesis. Finally, homology modeling and mutational analysis has revealed that the enzyme's catalytic triad contains a threonine residue (Thr117) instead of the usual serine.
authors.affiliation: Centre for Industrial Biotechnology and Biocatalysis, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, 9000, Gent, Belgium.
authors.forename: Koen
authors.initials: K
authors.lastname: Beerens
authors.forename: Wim
authors.initials: W
authors.lastname: Soetaert
authors.forename: Tom
authors.initials: T
authors.lastname: Desmet
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D020125:Mutation, Missense; D013379:Substrate Specificity; D013824:Thermus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D002238:Carbohydrate Epimerases; C100033:UDP acetylglucosamine-2-epimerase