Purification, crystallization and preliminary structural studies of dTDP-4-keto-6-deoxy-glucose-5-epimerase (EvaD) from Amycolatopsis orientalis, the fourth enzyme in the dTDP-L-epivancosamine biosynthetic pathway.
The vancomycin class of antibiotics is regarded as the last line of defence against Gram-positive bacteria. The compounds used clinically are very complex organic molecules and are made by fermentation. The biosynthesis of these is complex and fascinating. Its study holds out the prospect of utilizing genetic engineering of the enzymes in the pathway in order to produce novel vancomycin analogues. In part, this requires detailed structural insight into substrate specificity as well as the enzyme mechanism. The crystallization of one of the enzymes in the chloroeremomycin biosynthetic pathway (a member of the vancomycin family), dTDP-3-amino-4-keto 2,3,6-trideoxy-3-C-methyl-glucose-5-epimerase (EvaD) from Amycolatopsis orientalis, is reported here. The protein is fourth in the pathway which makes a carbohydrate essential for the activity of chloroeremomycin. The crystals of EvaD diffract to 1.5 A and have unit-cell parameters a = 98.6, b = 72.0, c = 57.1 A with space group P2(1)2(1)2. Data to this resolution were collected at the European Synchrotron Radiation Facility.
Merkel
Alexandra B
AB
The Centre for Biomolecular Sciences, The University, St Andrews KY16 9ST, Scotland.
Temple
Genevieve K
GK
Burkart
Michael D
MD
Losey
Heather C
HC
Beis
Kostantinos
K
Walsh
Christopher T
CT
Naismith
James H
JH
D000192:Actinomycetales / Q000201:enzymology*; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000302:isolation & purification; D002241:Carbohydrates / Q000737:chemistry; D018360:Crystallography, X-Ray / Q000379:methods*; D005947:Glucose / Q000031:analogs & derivatives* / Q000737:chemistry*; D008956:Models, Chemical; D009702:Nucleoside Diphosphate Sugars / Q000096:biosynthesis* / Q000737:chemistry; D019998:Racemases and Epimerases / Q000737:chemistry*; D013696:Temperature; D013942:Thymine Nucleotides / Q000096:biosynthesis* / Q000737:chemistry*
D002241:Carbohydrates; D009702:Nucleoside Diphosphate Sugars; D013942:Thymine Nucleotides; C112447:dTDP-6-deoxy-4-ketoglucose; C416082:thymine diphosphate epivancosamine; D019998:Racemases and Epimerases; D002238:Carbohydrate Epimerases; C470843:dTDP-4-keto-6-deoxyglucose-5-epimerase; D005947:Glucose