Expanding Pyrimidine Diphosphosugar Libraries Via Structure-Based Nucleotidylyltransferase Engineering

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 99; no. 21; pp. 13397 - 13402
• Main Authors: Barton, William A., Biggins, John B., Jiang, Jiqing, Thorson, Jon S., Nikolov, Dimitar B.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 15.10.2002; National Acad Sciences
• Subjects: Active sites; Amino Acid Substitution; Catalytic Domain - genetics; Chemistry; Crystallography, X-Ray; Design engineering; Drug Design; Enzymes; Escherichia coli - genetics; Glycosylation; Metabolism; Models, Molecular; Mutagenesis, Site-Directed; Nucleotides; Nucleotidyltransferases - genetics; Nucleotidyltransferases - metabolism; Oxygen; Phosphates; Physical Sciences; Promiscuity; Protein Engineering; Pyrimidine Nucleotides - biosynthesis; Pyrimidine Nucleotides - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Salmonella enterica - enzymology; Salmonella enterica - genetics; Structural engineering; Substrate Specificity; Sugar phosphates; Sugars
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.192468299

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 α-D-hexopyranosyl phosphates accepted by Salmonella enterica LT2 α-D-glucopyranosyl phosphate thymidylyltransferase (Ep). 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 α-D-hexopyranosyl phosphates accepted by Epto include α-D-allo-, α-D-altro-, and α-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.