Structure-function studies of two novel UDP-GlcNAc C6 dehydratases/C4 reductases. Variation from the SYK dogma.
Two subfamilies of UDP-GlcNAc C6 dehydratases were recently identified. FlaA1, a short soluble protein that exhibits a typical SYK catalytic triad, characterizes one of these subfamilies, and WbpM, a large membrane protein that harbors an altered SMK triad that was not predicted to sustain activity, represents the other subfamily. This study focuses on investigating the structure and function of these C6 dehydratases and the role of the altered triad as well as additional amino acid residues involved in catalysis. The significant activity retained by the FlaA1 Y141M triad mutant and the low activity of the WbpM M438Y mutant indicated that the methionine residue was involved in catalysis. A Glu(589) residue, which is conserved only within the large homologues, was shown to be essential for activity in WbpM. Introduction of this residue in FlaA1 enhanced the activity of the corresponding V266E mutant. Hence, this glutamate residue might be responsible for the retention of catalytic efficiency in the large homologues despite alteration of their catalytic triad. Mutations of residues specific for the short homologues (Asp(70), Asp(149)-Lys(150), Cys(103)) abolished the activity of FlaA1. Among them, C103M prevented dimerization but did not significantly affect the secondary structure. The fact that we could identify subfamily-specific residues that are essential for catalysis suggested an independent evolution for each subfamily of C6 dehydratases. Finally, the loss of activity of the FlaA1 G20A mutant provided evidence that a cofactor is involved in catalysis, and kinetic study of the FlaA1 H86A mutant revealed that this conserved histidine is involved in substrate binding. None of the mutations investigated altered the substrate, product, and function specificity of these enzymes.
Creuzenet
Carole
C
Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Urbanic
Robert V
RV
Lam
Joseph S
JS
D001426:Bacterial Proteins; D001665:Binding Sites; D002384:Catalysis; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers; D019281:Dimerization; D019075:Electrophoresis, Capillary; D004926:Escherichia coli / Q000201:enzymology; D006836:Hydro-Lyases / Q000737:chemistry* / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010088:Oxidoreductases / Q000737:chemistry* / Q000502:physiology*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D013997:Time Factors; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D017931:DNA Primers; C417241:FlaA1 protein, Helicobacter pylori; D010088:Oxidoreductases; D006836:Hydro-Lyases; D003545:Cysteine