Human glucose-6-phosphate dehydrogenase: the crystal structure reveals a structural NADP(+) molecule and provides insights into enzyme deficiency

Background: Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first co mmitted step in the pentose phosphate pathway; the generation of NADPH by t his enzyme is essential for protection against oxidative stress. The human enzyme is in a dimer<->tetramer equilibrium and its stability is dependent on NADP(+) concentration. G6PD deficiency results from many different point mutations in the X-linked gene encoding G6PD and is the most common human enzymopathy. Severe deficiency causes chronic non-spherocytic haemolytic an aemia; the usual symptoms are neonatal jaundice, favism and haemolytic anae mia. Results: We have determined the first crystal structure of a human G6PD (th e mutant Canton, Arg459-->Leu) at 3 Angstrom resolution. The tetramer is a dimer of dimers. Despite very similar dimer topology, there are two major d ifferences from G6PD of Leuconostoc mesenteroides: a structural NADP(+) mol ecule, close to the dimer interface but integral to the subunit, is visible in all subunits of the human enzyme; and an intrasubunit disulphide bond t ethers the otherwise disordered N-terminal segment. The few dimer-dimer con tacts making the tetramer are charge-charge interactions. Conclusions: The importance of NADP(+) for stability is explained by the st ructural NADP(+) site, which is not conserved in prokaryotes. The structure shows that point mutations causing severe deficiency predominate close to the structural NADP(+) and the dimer interface, primarily affecting the sta bility of the molecule. They also indicate that a stable dimer is essential to retain activity in vivo. As there is an absolute requirement for some G 6PD activity, residues essential for coenzyme or substrate binding are rare ly modified.