Genomic structure of GGH and CpG islands in the GGH promoter region. Two CpG islands in the GGH 5 promoter region extend into intron 1. The CpG1 and CpG2 regions (shaded areas) are indicated relative to the A of the translation start codon defined as nucleotide +1.

Structure modeling of wild-type and T127I variant GGH. (A) Comparison of local structure of wild-type GGH (green) versus the T127I variant (orange). The structure of T127I GGH model is superimposed onto that of wild-type GGH. The local backbone structures of open tail end, loop 123-128, 74-79 and beta 9 168-173, are depicted as green (GGH) or orange (T127I) ribbons. The side chain overlapping four residues from Cys124 to Thr127 is also represented in green (GGH) and orange (T127I). (B) The crystal structure of carbamoyl-phosphate synthetase (eCPS) variant H353N (magenta) and glutamine thioester (orange) complex was used to superimpose onto the complex of GGH (green) and MTXPG5 substrate by least-squares fitting of a-carbons of 20 residues around the active site. Docking models: (C) wild-type GGH with MTXPG5; (D) T127I variant with MTXPPG5; (E) wild-type GGH with MTXPG2; (F) T127I variant with MTXPG2. Docking models are shown at the end of the cleft-tail. The arrows indicate protrusions of the region at the surface of residues Cys124 and Leu125.

GGH activity in human leukemia cells is regulated by epigenetic modification, genetic polymorphisms and karyotypic abnormalities, which collectively determine interindividual differences in GGH activity and influence MTXPG accumulation in leukemia cells.