Abstract
Deficiency of NADH-cytochrome b5 reductase (cb5r) causes two clinically distinct phenotypes of recessive congenital methemoglobinemia (RCM). Type I patients often manifest cyanosis from birth, and in type II patients the cyanosis is accompanied by severe neurological impairment. The mechanisms responsible for the phenotypic differences between the two subgroups remain to be defined. The majority of patients harbor two different mutant alleles. To date 39 mutant variants of cb5r have been identified, 2 of which are common to both types of RCM. In order to characterize the individual cb5r variant proteins we have developed a novel heterologous expression system based on the structures of the rat and human proteins derived by X-ray crystallography. The system permits the investigation of the catalytic efficiencies, protein thermostability, FAD cofactor properties and substrate (NADH/NAD+) affinities of the variants. We have investigated four patients with type I RCM, one of whom was homozygous for the D239G mutation. The other three were compound heterozygous: R159-/D239G; G75S/V252M; and P275L/G291D, and one mutation, P275L, was novel. All patients showed reduced enzyme activity, in the range 0.5 to 5.8 IU/g Hb compared to normal values of 7.2 to 26.9 IU/g Hb. Individual variant proteins were prepared and the analytical data are summarised in the Table below.
Variant Catalytic Efficiency (% of normal) Thermal Stability (T50°C) NADH affinity (Km) NAD+ affinity (Ks) ND - not determined G75S 11 48 Normal 9-fold ↑ R159- 0 ND ND ND D239G 2 56 40-fold ↓ ND V252M 9 53 9-fold ↓ 18-fold ↑ E255- 0.4 51 100-fold ↓ ND P275L 0.2 53 437-fold ↓ ND G291D 43 49 1.3-fold ↓ 1.1-fold ↑ Wild type 100 57 normal normal
As expected all of the variants generated had decreased enzyme activity compared to wild type heterologous protein, supporting the validity of this approach. Thermal stability was decreased in the G75S, V252M and G291D variants. G75 is present in a highly conserved region in the FAD-binding lobe. Although it does not interact directly with the FAD prosthetic group it is important for association with cytochrome b5. Substitution of glycine at residue 75 by serine resulted in decreased enzyme activity and stability, with a marginal decrease in NADH affinity. The R159- variant protein was unstable and could not be isolated. Both the D239G and P275L mutations significantly reduced the affinity of cb5r for NADH, by 40-fold and 437-fold respectively. The rat cb5r model suggests that residue D239 is key for selecting between the NADPH and NADH pyridine nucleotides. This was confirmed by the 40-fold decrease in affinity for NADH and a 125-fold increase in affinity for NADPH. Residue P275 is located in a highly conserved region, which is important for the correct positioning and binding of NADH. Consequently, substitution of proline at 275 would affect the affinity of cb5r towards NADH, which was confirmed by the affinity constant measurements. These studies provide important information about the structure-function relationships of the variant cb5r proteins which may impart useful insights into the pathophysiological differences between type I and type II RCM.
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