Abstract
Diverse types of NDP-sugar pyrophosphorylase (NDP-sugar PPase) catalyze phosphosugars to form NDP-sugars, which serve as substrates for glycoconjugates synthesis. Among all prokaryotes analyzed, Anabaena contains the largest number of NDP-sugar PPase types, and can be a good model to understand the interaction and regulation of NDP-sugar PPases in a single organism. All NDP-sugar PPase genes from Anabaena were overexpressed in E. coli, the recombinant proteins were purified, and their enzymatic activities were determined. Enzyme alr2361CH1 and alr3400CH1, both were annotated as a GDP-Man PPase by phylogenetic analysis, were found to utilize GDP-Glc and UDP-Glc, respectively, as the preferred substrate. This finding is remarkable because this is the novel prokaryotic GDP-Glc PPase ever discovered. Both all3274CH1 and alr3400CH1 utilize UDP-Glc as the substrate, although the former exhibited a much higher activity. Interestingly, all3274CH1 is also capable of catalyzing an unusual activity - converting UDP-Gal into UTP and Gal 1-P independent of UDP-Glc. All NDP-sugar PPases in Anabaena requires magnesium ion for their maximum activities, and the optimal temperature for most NDP-PPase were at 47 °C except alr3400CH1. Moreover, pyruvate can serve as an allosteric effector and enhance the GDP-Glc PPase activity of alr2361CH1. To sum up, the characterization of the enzymatic properties of NDP-sugar PPases in Anabaena can provide critical information on the bacterial physiology and reveal previously unknown biological processes.