D-2HG mediates mutant Idh phenotypes in Drosophila. (A) Structure of active site of human (h)IDH1 (1T0L displayed with UCSF Chimera v1.5; National Institutes of Health)^64,65  with critical metal-binding residues shown, including Asp273, Asp275, and Asp279. Homology analysis shows that Asp334, Asp336, and Asp340 are the homologous residues in fly Idh. (B) D-2HG levels in pupae ubiquitously expressing no transgene, wild-type (WT) Idh, Idh-R163Q, Idh-R195H, and Idh-D>N in which the 3 metal-binding Asp residues are mutated to Asn (D334N-D336N-D340N), and expressing Idh containing the R195H gain-of-function mutation and the D334N-D336N-D340N metal-binding residue mutations (Idh-R195H-D>N). (C) Schematic showing NADPH-linked D-2HG production by Idh mutants, including Idh-R195H. D2HGDH is an FAD-linked enzyme that converts D-2HG to α-ketoglutarate. D2hgdh (CG3835) is a candidate D2HGDH homolog in Drosophila. (D) Pupae ubiquitously expressing Idh-R195H, D2hgdh, neither, or both were analyzed for D-2HG. Data are expressed as mean ± standard deviation from 2 replicates and are representative of 3 independent experiments.