Veronica M.S. Lam

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 committed step in the pentose phosphate pathway; the generation of NADPH by this 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 anaemia; the usual symptoms are neonatal jaundice, favism and haemolytic anaemia. RESULTS We have determined the first crystal structure of a human G6PD (the mutant Canton, Arg459-->Leu) at 3 A resolution. The tetramer is a dimer of dimers. Despite very similar dimer topology, there are two major differences from G6PD of Leuconostoc mesenteroides: a structural NADP(+) molecule, close to the dimer interface but integral to the subunit, is visible in all subunits of the human enzyme; and an intrasubunit disulphide bond tethers the otherwise disordered N-terminal segment. The few dimer-dimer contacts making the tetramer are charge-charge interactions. CONCLUSIONS The importance of NADP(+) for stability is explained by the structural 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 stability 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 G6PD activity, residues essential for coenzyme or substrate binding are rarely modified.

Structural studies of glucose-6-phosphate and NADP+ binding to human glucose-6-phosphate dehydrogenase

Human glucose-6-phosphate dehydrogenase (G6PD) is NADP(+)-dependent and catalyses the first and rate-limiting step of the pentose phosphate shunt. Binary complexes of the human deletion mutant, DeltaG6PD, with glucose-6-phosphate and NADP(+) have been crystallized and their structures solved to 2.9 and 2.5 A, respectively. The structures are compared with the previously determined structure of the Canton variant of human G6PD (G6PD(Canton)) in which NADP(+) is bound at the structural site. Substrate binding in DeltaG6PD is shown to be very similar to that described previously in Leuconostoc mesenteroides G6PD. NADP(+) binding at the coenzyme site is seen to be comparable to NADP(+) binding in L. mesenteroides G6PD, although some differences arise as a result of sequence changes. The tetramer interface varies slightly among the human G6PD complexes, suggesting flexibility in the predominantly hydrophilic dimer-dimer interactions. In both complexes, Pro172 of the conserved peptide EKPxG is in the cis conformation; it is seen to be crucial for close approach of the substrate and coenzyme during the enzymatic reaction. Structural NADP(+) binds in a very similar way in the DeltaG6PD-NADP(+) complex and in G6PD(Canton), while in the substrate complex the structural NADP(+) has low occupancy and the C-terminal tail at the structural NADP(+) site is disordered. The implications of possible interaction between the structural NADP(+) and G6P are considered.

What is the role of the second “structural” NADP+-binding site in human glucose 6-phosphate dehydrogenase?

Human glucose 6‐phosphate dehydrogenase, purified after overexpression in E. coli, was shown to contain one molecule/subunit of acid‐extractable “structural” NADP+ and no NADPH. This tightly bound NADP+ was reduced by G6P, presumably following migration to the catalytic site. Gel‐filtration yielded apoenzyme, devoid of bound NADP+ but, surprisingly, still fully active. Mr of the main component of “stripped” enzyme by gel filtration was ∼100,000, suggesting a dimeric apoenzyme (subunit Mr = 59,000). Holoenzyme also contained tetramer molecules and, at high protein concentration, a dynamic equilibrium gave an apparent intermediate Mr of 150 kDa. Fluorescence titration of the stripped enzyme gave the K d for structural NADP+ as 37 nM, 200‐fold lower than for “catalytic” NADP+. Structural NADP+ quenches 91% of protein fluorescence. At 37°C, stripped enzyme, much less stable than holoenzyme, inactivated irreversibly within 2 d. Inactivation at 4°C was partially reversed at room temperature, especially with added NADP+. Apoenzyme was immediately active, without any visible lag, in rapid‐reaction studies. Human G6PD thus forms active dimer without structural NADP+. Apparently, the true role of the second, tightly bound NADP+ is to secure long‐term stability. This fits the clinical pattern, G6PD deficiency affecting the long‐lived non‐nucleate erythrocyte. The K d values for two class I mutants, G488S and G488V, were 273 nM and 480 nM, respectively (seven‐ and 13‐fold elevated), matching the structural prediction of weakened structural NADP+ binding, which would explain decreased stability and consequent disease. Preparation of native apoenzyme and measurement of K d constant for structural NADP+ will now allow quantitative assessment of this defect in clinical G6PD mutations.

Solution of the structure of tetrameric human glucose 6-phosphate dehydrogenase by molecular replacement

Recombinant human glucose 6-phosphate dehydrogenase (G6PD) has been crystallized and its structure solved by molecular replacement. Crystals of the natural mutant R459L grow under similar conditions in space groups P212121 and C2221 with eight or four 515-residue molecules in the asymmetric unit, respectively. A non-crystallographic 222 tetramer was found in the C2221 crystal form using a 4 A resolution data set and a dimer of the large beta + alpha domains of the Leuconostoc mesenteroides enzyme as a search model. This tetramer was the only successful search model for the P212121 crystal form using data to 3 A. Crystals of the deletion mutant DeltaG6PD grow in space group F222 with a monomer in the asymmetric unit; 2.5 A resolution data have been collected. Comparison of the packing of tetramers in the three space groups suggests that the N-terminal tail of the enzyme prevents crystallization with exact 222 molecular symmetry.

Glucose 6-phosphate dehydrogenase (G6PD) deficiency in elderly Chinese women heterozygous for G6PD variants

deficiencies [Yoshida et al., 1971]. Although chronic hemolysis is absent, oxidative hemolysis caused by drugs and infections may occur [Beutler, 1994]. Clinically overt G6PD deficiency in females is rare and is mostly attributed to homozygosity or compound heterozygosity for G6PDvariants. Other rare causes included the 45,X genotype, clonal hematopoeisis [Au et al., 2002] or extreme X chromosome inactivation [Beutler, 1994]. We describe two elderly Chinese womenwith severe hemolysis TABLE I. Index Patients, G6PD Deficient Females, and Their Family Members

Molecular characterization of β‐globin gene mutations in patients with β‐thalassaemia intermedia in South China

We have studied the spectrum of mutations producting β‐thalassaemia intermedia in South China. The methods of mutation detection include oligonucleotide analysis, polymerase chain reaction amplification of the β‐globin gene and direct genomic sequencing. The mutations have been identified in 22 β‐globin genes from the patients in 11 unrelated families. Seven different mutations have been identified and the A to G substitution in the TATA box of the β‐globin gene accounts for 42% of these mutant β‐globin genes. Most patients have a β+ thalassaemia and one copy of the TATA box mutation. In two patients with β) thalassaemia intermedia the mild phenotype may be explained in one by the presence of the ‐ + ‐ + + 5’β‐globin gene cluster haplotype which contains the Xmn I site ‐158 nt to the Gγ‐globin gene or in the other by the number of α‐globin genes present.

Rapid detection of common Chinese glucose-6-phosphate dehydrogenase (G6PD) mutations by denaturing gradient gel electrophoresis (DGGE).

We describe here the use of denaturing gradient gel electrophoresis (DGGE) to detect the most common Chinese glucose-6-phosphate dehydrogenase (G6PD) variants, which are the single point mutations: G-->T at nt 1376, G-->A at 1388 both in exon 12 and A-->G at nt 95 in exon 02. In each case, the mutant allele resolves well from the normal allele(s). The distinct heteroduplex bands are characteristic of a particular genotype suggesting that this feature is very useful for identifying all heterozygous carriers for this and other X-linked diseases. When the analysis is extended to other exons, DGGE scans the gene and coupled with direct sequencing, it leads to the identification of new G6PD variation(s). With this approach, we identified a mutation in exon 9 which had not been reported in Hong Kong. Since DGGE can rapidly screen many unknown samples in one gel, this approach could be used to diagnose these G6PD mutations and to identify the at-risk for counselling.

Two new rat α-globin sequences as identified by the conserved region PCR

Based on the notion that regions of structural genes which encode critical domains of the corresponding proteins are highly conserved among closely related species, oligonucleotide primers were designed and used to amplify the α-globin sequence(s) of the Sprague-Dawley (SD) rat. Data of these amplified sequence constructs showed that two new rat a-globin specific sequences have been identified. Southern hybridization confirmed the presence of these sequences in the rat genome.