Structure, mechanism and engineering of a nucleotidylyltransferase as a first step toward glycorandomization.
Metabolite glycosylation is affected by three classes of enzymes: nucleotidylyltransferases, which activate sugars as nucleotide diphospho-derivatives, intermediate sugar-modifying enzymes and glycosyltransferases, which transfer the final derivatized activated sugars to aglycon substrates. One of the first crystal structures of an enzyme responsible for the first step in this cascade, alpha-D-glucopyranosyl phosphate thymidylyltransferase (Ep) from Salmonella, in complex with product (UDP-Glc) and substrate (dTTP) is reported at 2.0 A and 2.1 A resolution, respectively. These structures, in conjunction with the kinetic characterization of Ep, clarify the catalytic mechanism of this important enzyme class. Structure-based engineering of Ep produced modified enzymes capable of utilizing 'unnatural' sugar phosphates not accepted by wild type Ep. The demonstrated ability to alter nucleotidylyltransferase specificity by design is an integral component of in vitro glycosylation systems developed for the production of diverse glycorandomized libraries.
Barton
W A
WA
Cellular Biochemistry and Biophysics Program, Joan and Sanford I. Weill Graduate School of Medical Sciences, Cornell University, 1275 York Avenue, New York 10021, USA.
Lesniak
J
J
Biggins
J B
JB
Jeffrey
P D
PD
Jiang
J
J
Rajashankar
K R
KR
Thorson
J S
JS
Nikolov
D B
DB
D001665:Binding Sites; D002384:Catalysis; D002413:Cations, Divalent / Q000378:metabolism; D018360:Crystallography, X-Ray; D006031:Glycosylation; D016695:Glycosyltransferases / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019779:Salmonella enterica / Q000201:enzymology*; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D002413:Cations, Divalent; D019151:Peptide Library; D013942:Thymine Nucleotides; D016695:Glycosyltransferases; D009713:Nucleotidyltransferases; C020587:glucose-1-phosphate thymidylyltransferase; C024157:thymidine 5'-triphosphate; D014532:Uridine Diphosphate Glucose