Modeling human glucose-6-phosphate dehydrogenase mutations using C. elegans GSPD-1

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked, recessive condition that causes intermittent jaundice or hemolytic anemia because of low NADPH levels in red blood cells. We performed steady-state enzyme kinetics with the recombinant C. elegans ortholog of human G6PD, GSPD-1, and two mutants containing amino acid changes found in human patients. The K _M values for glucose-6-phosphate were 100 ± 27 µM, 80 ± 22 µM, and 1000 ± 300 µM for the wild-type, D60N, and R252L GSPD-1 enzymes, respectively. The specific activities of the D60N and R252L mutants were 59% and 11%, respectively, of the wild-type value. Protein homology modeling suggested that the R252L mutation was more severe because the mutation caused a shift in the position of some active site residues. The D60N mutation may have affected the conformation of an outer loop of the enzyme. These data demonstrate that GSPD-1 is a promising model for human G6PD deficiencies, with the advantage that potential treatments could be studied in vivo in C. elegans.

Figure 1. The D60N and R252L mutations in GSPD-1 reduce enzyme activity, and the R252L mutation appears to alter the position of the active site residues

(A) The protein sequence alignments of C. elegans GSPD-1 (top line) and human G6PD (bottom line) surrounding the two mutation sites (underlined) are shown. The middle line of text shows identical amino acids, and a + symbol indicates a conservative substitution. The numbers show the amino acid positions at the ends of each sequence. (B) Michaelis-Menten graph of enzyme velocities (in μmol/min/mg or U/mg) for wild-type GSPD-1 (blue circles), the D60N mutant (green triangles), and the R252L mutant (purple squares) at varying glucose-6-phosphate (G6P) concentrations. All of the data points are shown, and the lines indicate the best-fit line. (C) This table shows the results from the Michaelis-Menten fits shown in B with standard errors. (D) View of the active site in GSPD-1 models. The wild-type structure is shown in beige, and the R252L structure is shown in light blue. Asp264 and His269 bind to the glucose moiety of G6P, His207 and Tyr208 bind to the phosphate moiety of G6P, and Arg252/Leu252 are at the site of the mutation. The positions of these amino acids in each structural model are shown.