Human NADH-cytochrome B5 Reductase Research Articles

Plant polyphenols as electron donors for erythrocyte plasma membrane redox system: validation through in silico approach

BackgroundThe plasma membrane redox system (PMRS) has extensively been studied in erythrocytes. The PMRS plays an important role in maintaining plasma redox balance and provides a protective mechanism against oxidative stress. Earlier it was proposed that only NADH or NADPH provided reducing equivalents to PMRS; however, now it is acknowledged that some polyphenols also have the ability to donate reducing equivalents to PMRS.MethodsTwo different docking simulation softwares, Molegro Virtual Docker and Glide were used to study the interaction of certain plant polyphenols viz. quercetin, epigallocatechin gallate, catechin epicatechin and resveratrol with human erythroyte NADH-cytochrome b5 reductase, which is a component of PMRS and together with the identification of minimum pharmacophoric feature using Pharmagist.ResultsThe derived common minimum pharmacophoric features show the presence of minimum bioactive component in all the selected polyphenols. Our results confirm wet lab findings which show that these polyphenols have the ability to interact and donate protons to the Human NADH-cytochrome b5 reductase.ConclusionWith the help of these comparative results of docking simulation and pharmacophoric features, novel potent molecules can be designed with higher efficacy for activation of the PMRS system.

An enhanced promoter trap protocol.

An enhanced promoter trap protocol.

Role of Lys-110 of human NADH-cytochrome b5 reductase in NADH binding as probed by site-directed mutagenesis

Lys-110 of human NADH-cytochrome b 5 reductase was replaced by Ala, Met, or Arg by site-directed mutagenesis to evaluate the role of the residue. K m values of purified Lys-110 → Ala and Lys-110 → Met mutants for NADH were approximately 200-fold and 1,100-fold higher than that of the wild-type, respectively, while the value of the Arg mutant was almost the same as that of the wild-type. These results indicate that the positive charge at position 110 is important for NADH binding. The k cat value of Lys-110 → Ala was not affected, indicating that the residue only participates in the binding process in the reaction by forming an ionic interaction with phosphoryl group of NADH.

Role of cysteine residues in human NADH-cytochrome b5 reductase studied by site-directed mutagenesis. Cys-273 and Cys-283 are located close to the NADH-binding site but are not catalytically essential.

Human NADH-cytochrome b5 reductase (EC 1.6.2.2) contains 4 cyteine residues (Cys-203, -273, -283, and -297). Cys-283 was previously proposed to be involved in NADH binding by chemical modification (Hackett, C. S., Novoa, W. B., Ozols, J., and Strittmatter, P. (1986) J. Biol. Chem. 261, 9854-9857). In the present study the role of cysteines in the enzyme was probed by replacing these residues by Ser, Ala, or Gly employing site-directed mutagenesis and chemical modification. Four mutants, in which 1 of the 4 Cys residues was replaced by Ser, retained comparable kcat and Km values to those of the wild type. All of these mutants were as sensitive as the wild type to treatment with SH modifiers, while a double mutant, C273S/C283S was resistant. Since inhibition by SH modifiers was protected by NADH, Cys-273 and Cys-283 were implicated to be close to the NADH-binding site. C273A and C273A/C283A mutants showed approximately one-fifth of the enzyme-FAD reduction rate of the wild type as revealed by steady-state kinetics and by stopped-flow analysis. Anaerobic titration has shown that reduction and re-oxidation processes including formation of the red semiquinone of these mutants were not significantly altered from those of the wild type. From these results it was concluded that none of the Cys residues of the enzyme are essential in the catalytic reaction, but Cys-273 conserved among the enzymes homologous to NADH-cytochrome b5 reductase homologous to NADH-cytochrome b5 reductase plays role(s) in facilitating the reaction. A difference spectrum with a peak at 317 nm, which was formerly considered to be derived from the interaction between NAD+ and Cys-283 of the reduced enzyme, appeared upon binding of NAD+ not only to the reduced wild type enzyme but also to the C273A/C283A mutant in which both of the Cys residues close to the NADH-binding site were replaced.

Structural role of serine 127 in the NADH-binding site of human NADH-cytochrome b5 reductase.

Serine 127 of human NADH-cytochrome b5 reductase was replaced by proline and alanine by site-directed mutagenesis. The former mutation has been found in the genes of patients with hereditary deficiency of the enzyme. Both the mutant enzymes (Ser-127----Pro mutant and Ser-127----Ala mutant) were overproduced in Escherichia coli and purified to homogeneity. The two purified mutant enzymes showed indistinguishable spectral properties which differed from those of the wild-type enzyme. The mutant enzymes showed higher molecular extinction coefficients at 462 nm than that of the wild-type enzyme. Quenching of FAD fluorescence in these mutant enzymes was significantly less than that in the wild-type enzyme. Furthermore, circular dichroism spectra of the mutant enzymes were different, in both the visible and ultraviolet regions, from that of the wild-type enzyme. The spectra of the mutant enzymes in the visible region were restored to almost the same spectrum as the wild type upon reduction with NADH. Ser-127----Pro mutant and Ser-127----Ala mutant showed very low Kcat/Km (NADH) values (5 x 10(7) and 3.5 x 10(7) s-1 M-1, respectively) with cytochrome b5 as an electron acceptor, than that of the wild-type enzyme (Kcat/Km (NADH) = 179 x 10(7) s-1 M-1), while the Kcat/Km (cytochrome b5) value for each enzyme was similar. The mutant enzymes were less thermostable than the wild-type enzyme. These results indicate that serine 127 plays an important role to maintain the structure of the NADH-binding site in the enzyme.

Isolation and characterization of the nitrate reductase structural gene of Chlamydomonas reinhardtii.

The nitrate reductase structural gene of Chlamydomonas reinhardtii has been isolated from a genomic library by using a nitrate reductase cDNA probe from barley. Restriction fragment length polymorphism analyses mapped the Chlamydomonas clone (B6a) to the nitrate reductase structural gene locus nit-1. Overlapping inserts cover a region of the genome of about 24 kilobases containing the entire gene, which spans approximately 5-8 kilobases. Sequence analysis of DNA fragments from the B6a clone demonstrated a high degree of sequence similarity at the amino acid level with regions corresponding to portions of the heme and FAD/NADH-binding domains of tobacco and Arabidopsis thaliana nitrate reductases and human NADH cytochrome b5 reductase. The identity of the cloned gene as nitrate reductase was confirmed by its ability to complement a nit-1 mutation upon transformation. The nitrate reductase gene produced a 3.4-kilobase transcript in cells derepressed with nitrate; the transcript was undetectable in cells grown in the presence of ammonium. In cells that contain a mutation in the putative regulatory gene nit-2, significantly lower levels of the 3.4-kilobase transcript were found, indicating that the wild-type nit-2 gene is involved in the control of nitrate reductase transcript levels.

The organization and the complete nucleotide sequence of the human NADH-cytochrome b5 reductase gene

The organization and the complete nucleotide (nt) sequence of the b5R gene encoding human NADH-cytochrome b5 reductase (b5R; EC 1.6.2.2) have been determined by a combination of restriction mapping and nt sequence analysis of overlapping genomic DNA clones. The entire gene is about 31 kb in length and contains nine exons and eight introns. Exon 2 contains the junction of the membrane-binding domain and the catalytic domain of b5R, indicating that two forms of b5R, a soluble and a membrane-bound form, are generated by post-translational processing. The 5' portion of the b5R gene lacks the canonical 5' transcriptional regulatory elements, but contains five copies of the GC box sequence G-G-G-C-G-G. While the average G + C content of the b5R gene is 55%, that of the 5' portion of the gene is extraordinarily high (86%). The CpG dinucleotide sequence was found at a very high frequency in this G + C-rich region. These structural features are very similar to those of the regulatory regions of constitutively expressed ‘housekeeping’ genes. Several transcription start points were identified by the primer extension experiment. Seventeen complete and twelve incomplete Alu family sequences were found in introns. An uncanonical polyadenylation signal was detected in the 3'-untranslated region of the gene as A-G-T-A-A-A instead of A-A-T-A-A-A.