GMPPB mutants exhibit significantly decreased enzymatic activity. a-b Validation of the designed system for evaluating GMPPB’s enzymatic activity. Absence of Mg2+ or substrate mannose-1-p leads to no detectable formation of GDP-mannose (a). The time course of GDP-man production catalyzed by variant doses of GMPPB (b). 0 μg, 5 μg, 10 μg or 20 μg purified GMPPB was used to catalyze the reaction, which was terminated at the indicated time. The reaction mixtures were subjected to Q-ion exchange chromatography, and the amount of GDP-mannose was determined. c V111G mutation causes significantly decreased activity of GMPPB. Purified GMPPB WT, GMPPB V111G, or GMPPB G214S were subjected to activity detection. Compared to the WT, V111G mutation caused significant reduction of GMPPB activity, whereas GMPPB G214 variant retained activity comparable to WT. d Disease-associated GMPPB mutation results in significantly decreased enzymatic activity. 17 reported GMPPB mutants identified in patients were expressed in E.coli and subjected to activity detection. All the tested mutants exhibited significantly compromised activity relative to GMPPB WT. Mean ± SD, ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05. p values were calculated using one-way ANOVA, Tukey’s multiple comparisons test
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