Interaction of NADP+ and NAD-capped RNA with Wildtype and Mutant Human Glucose-6-Phosphate Dehydrogenase

Kali Rivera, “Interaction of NADP+ and NAD-capped RNA with Wildtype and Mutant Human Glucose-6-Phosphate Dehydrogenase” 

Mentor: David Frick, Chemistry & Biochemistry, Letters & Science (College of) 

Poster #133 

The recent discovery that RNA polymerases frequently initiate synthesis with co-enzymes like nicotinamide adenine dinucleotide (NAD+), which then serve as atypical cap-like structures, has inspired the search for enzymes that might use NAD-capped RNA as a substrate. One possible candidate is human glucose-6-phosphate dehydrogenase (hG6PD) because hG6PD has two distinct NADP+ bindings. One site binds the catalytic NADP+ adjacent to glucose-6 phosphate (G6P). The second site is far from G6P, and the bound “structural” NADP+ has been proposed to stabilize the G6PD oligomer. Humans have also evolved numerous mutations in the hG6PD gene linked to G6PD deficiency disease, and many disease-linked mutations map near the NADP+ binding sites. Here, we examine the effects of dinucleotides and NAD-capped RNA on purified human G6PD encoded by the wild type, the “Wisconsin” or the “Chile” G6PD deficiency alleles. In the Wisconsin allele, Arg393 near the structural NADP+ site is replaced by glycine, destabilizing the enzyme’s structure. Arg198 is replaced by proline in the Chile allele, disrupting the active site. Genes encoding all three hG6PD proteins were cloned into pET28a. After the recombinant His-tagged proteins were purified using nickel-affinity columns, reactions catalyzed by each enzyme were monitored using NADPH absorbance, NADPH fluorescence, or by coupling reactions to diaphorase and monitoring the reduction of resazurin to resorufin. Differences were observed between the wild-type and mutant enzymes, but reduction of NAD-capped RNA was not observed with any protein. Some inhibition of G6PD-catalyzed reactions by NAD-capped RNA was observed with wildtype and Chile alleles. However, less inhibition was observed with the Wisconsin allele. These preliminary results suggest that the G6PD structural NADP+ binding site might accommodate NAD-capped RNA.