title: Elucidation of substrate specificity in Aspergillus nidulans UDP-galactose-4-epimerase.
abstract: The frequency of invasive fungal infections has rapidly increased in recent years. Current clinical treatments are experiencing decreased potency due to severe host toxicity and the emergence of fungal drug resistance. As such, new targets and their corresponding synthetic pathways need to be explored for drug development purposes. In this context, galactofuranose residues, which are employed in fungal cell wall construction, but are notably absent in animals, represent an appealing target. Herein we present the structural and biochemical characterization of UDP-galactose-4-epimerase from Aspergillus nidulans which produces the precursor UDP-galactopyranose required for galactofuranose synthesis. Examination of the structural model revealed both NAD(+) and UDP-glucopyranose were bound within the active site cleft in a near identical fashion to that found in the Human epimerase. Mutational studies on the conserved catalytic motif support a similar mechanism to that established for the Human counterpart is likely operational within the A. nidulans epimerase. While the K m and k cat for the enzyme were determined to be 0.11 mM and 12.8 s(-1), respectively, a single point mutation, namely L320C, activated the enzyme towards larger N-acetylated substrates. Docking studies designed to probe active site affinity corroborate the experimentally determined activity profiles and support the kinetic inhibition results.
authors.affiliation: Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors.forename: Sean A
authors.initials: SA
authors.lastname: Dalrymple
authors.forename: John
authors.initials: J
authors.lastname: Ko
authors.forename: Inder
authors.initials: I
authors.lastname: Sheoran
authors.forename: Susan G W
authors.initials: SG
authors.lastname: Kaminskyj
authors.forename: David A R
authors.initials: DA
authors.lastname: Sanders
mesh_terms: D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000201:enzymology* / Q000235:genetics; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014534:UDPglucose 4-Epimerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014532:Uridine Diphosphate Glucose / Q000737:chemistry / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D009243:NAD; D014534:UDPglucose 4-Epimerase; D014532:Uridine Diphosphate Glucose