Structure-based engineering of E. coli galactokinase as a first step toward in vivo glycorandomization.
In vitro glycorandomization is a rapid chemoenzymatic strategy to diversify complex natural product scaffolds. The glycorandomization sugar activation pathway is dependent upon the efficient construction of diverse sugar-1-phosphate libraries. In the context of the previously evolved GalK Y371H "gatekeeper" mutation, the active site M173L mutation described herein presents a kinase with remarkably broadened substrate range to include 28 diverse natural and unnatural sugars. Among these new substrates, 6-azido-6-deoxy-galactose and 6-azido-6-deoxy-glucose present unique chemical probes to assess the utility of an E. coli Y371H/M173L-GalK-overproducing strain to generate unnatural sugar-1-phosphates in vivo. Remarkably, the in vivo conversion of both unnatural sugars rival that demonstrated in vitro. This notable in vivo success stands as the first step toward constructing short sugar-activation pathways in vivo and, ultimately, in vivo natural-product glycorandomization.
Laboratory for Biosynthetic Chemistry, Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, 777 Highland Avenue, Madison, WI 53705, USA.
Jie
J
Yang
Xun
X
Fu
Jianchun
J
Liao
Lesley
L
Liu
Jon S
JS
Thorson
D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005686:Galactokinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011897:Random Allocation; D013379:Substrate Specificity
D002241:Carbohydrates; D005686:Galactokinase