Expanding pyrimidine diphosphosugar libraries via structure-based nucleotidylyltransferase engineering.
In vitro "glycorandomization" is a chemoenzymatic approach for generating diverse libraries of glycosylated biomolecules based on natural product scaffolds. This technology makes use of engineered variants of specific enzymes affecting metabolite glycosylation, particularly nucleotidylyltransferases and glycosyltransferases. To expand the repertoire of UDP/dTDP sugars readily available for glycorandomization, we now report a structure-based engineering approach to increase the diversity of alpha-d-hexopyranosyl phosphates accepted by Salmonella enterica LT2 alpha-d-glucopyranosyl phosphate thymidylyltransferase (E(p)). This article highlights the design rationale, determined substrate specificity, and structural elucidation of three "designed" mutations, illustrating both the success and unexpected outcomes from this type of approach. In addition, a single amino acid substitution in the substrate-binding pocket (L89T) was found to significantly increase the set of alpha-d-hexopyranosyl phosphates accepted by E(p) to include alpha-d-allo-, alpha-d-altro-, and alpha-d-talopyranosyl phosphate. In aggregate, our results provide valuable blueprints for altering nucleotidylyltransferase specificity by design, which is the first step toward in vitro glycorandomization.
Barton
William A
WA
Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Biggins
John B
JB
Jiang
Jiqing
J
Thorson
Jon S
JS
Nikolov
Dimitar B
DB
D019943:Amino Acid Substitution; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D015195:Drug Design; D004926:Escherichia coli / Q000235:genetics; D006031:Glycosylation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D011742:Pyrimidine Nucleotides / Q000096:biosynthesis / Q000737:chemistry*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019779:Salmonella enterica / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D011742:Pyrimidine Nucleotides; D011994:Recombinant Proteins; D009713:Nucleotidyltransferases; C020587:glucose-1-phosphate thymidylyltransferase