Two site-directed mutations are required for the conversion of a sugar dehydratase into an aminotransferase.
L-colitose and d-perosamine are unusual sugars found in the O-antigens of some Gram-negative bacteria such as Escherichia coli, Vibrio cholerae, and Salmonella enterica, among others. The biosynthetic pathways for these two sugars begin with the formation of GDP-mannose from d-mannose 1-phosphate and GTP followed by the subsequent dehydration and oxidation of GDP-mannose to yield GDP-4-keto-6-deoxymannose. Following the production of GDP-4-keto-6-deoxymannose, the two pathways diverge. In the case of GDP-perosamine biosynthesis, the next step involves an amination reaction at the C-4' position of the sugar, whereas in GDP-colitose production, the 3'-hydroxyl group is removed. The enzymes catalyzing these reactions are GDP-perosamine synthase and GDP-4-keto-6-deoxymannose-3-dehydratase (ColD), respectively. Both of these enzymes are pyridoxal 5'-phosphate (PLP) dependent, and their three-dimensional structures place them into the well-characterized aspartate aminotransferase superfamily. A comparison of the active site architecture of ColD from E. coli (strain 5a, type O55:H7) to that of GDP-perosamine synthase from Caulobacter crescentus CB15 suggested that only two mutations would be required to convert ColD into an aminotransferase. Here we present a combined structural and functional analysis of the ColD S187N/H188K mutant protein that, indeed, has been converted from a sugar dehydratase into an aminotransferase.
Cook
Paul D
PD
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Kubiak
Rachel L
RL
Toomey
Daniel P
DP
Holden
Hazel M
HM
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000378:metabolism; D016935:Caulobacter crescentus / Q000201:enzymology / Q000378:metabolism; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006155:Guanosine Diphosphate Mannose / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D013379:Substrate Specificity; D000637:Transaminases / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C054686:GDP-4-keto-6-deoxymannose; D006155:Guanosine Diphosphate Mannose; D000637:Transaminases; C526462:GDP-4-keto-6-deoxymannose-3-dehydratase, E coli; D006836:Hydro-Lyases; D002238:Carbohydrate Epimerases; C061272:perosamine synthetase