Solution structure of the B form of oxidized rat microsomal cytochrome b5 and backbone dynamics via 15N rotating-frame NMR-relaxation measurements. Biological implications.
Cytochrome b5 in solution has two isomers (A and B) differing by a 180 degrees rotation of the protoporphyrin IX plane around the axis defined by the alpha and gamma meso protons. Homonuclear and heteronuclear NMR spectroscopy has been employed in order to solve the solution structure of the minor (B) form of the oxidized state of the protein and to probe its backbone dynamics in the microsecond--ms timescale in both oxidation states. A family of 40 conformers has been obtained using 1302 meaningful NOEs and 220 pseudocontact shifts and is characterized by high quality and good resolution (rmsd to the mean structure of 0.055 +/- 0.009 nm and 0.103 +/- 0.011 nm for backbone and heavy atoms, respectively). Extensive comparisons of the structural and dynamics changes associated with the A-to-B form interconversion for both oxidation states were subsequently performed. Propionate 6 experiences a redox-state-dependent reorientation as does propionate 7 in the A form. Significant insights are obtained into the role of the protein frame for efficient biological function and backbone mobility is proposed to be one of the factors that could control the reduction potential of the heme. (+info
Association of cytochrome b5 with 16-androstene steroid synthesis in the testis and accumulation in the fat of male pigs.
The 16-androstene steroids, one of the principal causes of boar taint, are synthesized in the testis by the andien-beta synthase enzyme system. This system has been shown in vitro to involve both cytochrome P450c17 and cytochrome b5. The objective of this work was to investigate the relationship between the levels of cytochrome b5 in the testis, in vitro steroidogenesis, and the accumulation of 16-androstene steroids in the fat of pubertal boars. We found that the in vitro rate of 16-androstene steroidogenesis in testis microsomes was correlated with 16-androstene steroid concentrations in fat (r = .66, P < .01). Western blots were used to determine the amounts of cytochrome b5 and cytochrome P450c17 protein in testis, and two immunoreactive cytochrome b5 proteins of approximately 12 and 16 kDa were found. Levels of cytochrome P450c17 or the high molecular weight cytochrome b5 in testis were not significantly correlated to levels of 16-androstene steroids in fat. However, levels of total cytochrome b5 immunoreactive protein and levels of the low molecular weight immunoreactive cytochrome b5 were correlated to fat 16-androstene steroid concentrations (r = .59, P < .001; r = .72, P = .0001, respectively). Levels of the low molecular weight immunoreactive cytochrome b5 were also correlated to 16-androstene steroid synthesis rates in vitro (r = .62, P < .05). These results indicate that increased levels of a low molecular weight immunoreactive cytochrome b5 protein, and not of cytochrome P450c17, are related to increased testicular 16-androstene steroid production and accumulation in fat. These results support the hypothesis that selection for reduced levels of this low molecular weight immunoreactive cytochrome b5 protein in the testis may result in decreased levels of 16-androstene steroids in fat and reduced boar taint in uncastrated male pigs. (+info
Genetic analysis of cytochrome b5 from arachidonic acid-producing fungus, Mortierella alpina 1S-4: cloning, RNA editing and expression of the gene in Escherichia coli, and purification and characterization of the gene product.
Information on the amino acid sequences of the internal peptide fragments of cytochrome b5 from Mortierella hygrophila was used to prepare synthetic oligonucleotides as primers for the polymerase chain reaction. A 100-base DNA fragment was thus amplified, by using a genomic gene from Mortierella alpina 1S-4 as a template, which produced polyunsaturated fatty acids such as arachidonic acid. The amplified DNA fragment was used as the probe to clone both a 523-base cDNA fragment and a 2.1-kilobase SalI-NruI genomic fragment coding for the whole M. alpina 1S-4 cytochrome b5. On the basis of nucleotide sequences of both cytochrome b5 genomic gene and cDNA, the genomic cytochrome b5 gene was found to consist of four exons and three introns. A novel type of RNA editing, in which the cDNA included either guanine insertion or adenine-->guanine substitution at one base upstream of poly(A), was interestingly observed. The deduced amino acid sequence of M. alpina 1S-4 cytochrome b5 showed significant similarities with those of cytochrome b5s from other organisms such as rat, chicken, and yeast. The soluble form of the cytochrome b5 gene was expressed to 16% of the total soluble protein in Escherichia coli. The holo-cytochrome b5 accounted for 8% of the total cytochrome b5 in the transformants. The purified cytochrome b5 showed the oxidized and reduced absorbance spectra characteristic of fungal microsomal cytochrome b5. (+info
Lysine mutagenesis identifies cationic charges of human CYP17 that interact with cytochrome b5 to promote male sex-hormone biosynthesis.
Human CYP17 (17alpha-hydroxylase-17,20-lyase; also cytochrome P450c17 or cytochrome P450(17alpha)) catalyses a hydroxylation reaction and another reaction involving the cleavage of a C-C bond (the lyase activity) that is required only for androgen production. Single amino acid mutations in human CYP17, Arg(347)-->His and Arg(358)-->Gln, have been reported to result in the loss of the lyase activity and to cause sexual phenotypic changes in 46XY male patients. By using site-directed mutagenesis we show here that another mutation in human CYP17, Arg(449)-->Ala, for which human variants have yet not been described, also leads to selective lyase deficiency. Furthermore, all the three types of mutants display a loss of responsiveness to cytochrome b(5), an interaction that is essential for lyase activity, and hence male sex-hormone biosynthesis. That the defect could be essentially reversed by lysine mutagenesis has led to the conclusion that the cationic charges on all three residues (at the positions of Arg(347), Arg(358), Arg(449)) are vital for the functional interaction of CYP17 with cytochrome b(5) and that the loss of any one of these cationic charges is catastrophic. (+info
Enhancement of cytochrome P-450 3A4 catalytic activities by cytochrome b(5) in bacterial membranes.
Activities of testosterone, nifedipine, and midazolam oxidation by recombinant cytochrome P-450 (P-450) 3A4 coexpressed with human NADPH-P-450 reductase (NPR) in bacterial membranes (CYP3A4/NPR membranes) were determined in comparison with those of other recombinant systems and of human liver microsomes with high contents of CYP3A4. Growth conditions for Escherichia coli transformed with the bicistronic construct affected expression levels of CYP3A4 and NPR; an excess of NPR over P-450 in membrane preparations enhanced CYP3A4-dependent testosterone 6beta-hydroxylation activities of the CYP3A4/NPR membranes. Cytochrome b(5) (b(5)) and apolipoprotein b(5) further enhanced the testosterone 6beta-hydroxylation activities of CYP3A4/NPR membranes after addition to either bacterial membranes or purified enzymes. NPR was observed to enhance catalytic activity when added to the CYP3A4/NPR membranes, either in the form of bacterial membranes or as purified NPR (in combination with cholate and b(5)). Apparent maximal activities of testosterone 6beta-hydroxylation in CYP3A4/NPR membranes were obtained when the molar ratio of CYP3A4/NPR/b(5) was adjusted to 1:2:1 by mixing membranes containing each protein. Testosterone 6beta-hydroxylation, nifedipine oxidation, and midazolam 4- and 1'-hydroxylation activities in CYP3A4/NPR membranes plus b(5) systems were similar to those measured with microsomes of insect cells coexpressing CYP3A4 with NPR and/or of human liver microsomes, based on equivalent CYP3A4 contents. These results suggest that CYP3A4/NPR membrane systems containing b(5) are very useful models for prediction of the rates for liver microsomal CYP3A4-dependent drug oxidations. (+info
Histidine-41 of the cytochrome b5 domain of the borage delta6 fatty acid desaturase is essential for enzyme activity.
Unlike most other plant microsomal desaturases, the Delta6-fatty acid desaturase from borage (Borago officinalis) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b5. To determine if this domain serves as a functional electron donor for the Delta6-fatty acid desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Delta6-fatty acid desaturase resulted in the synthesis and accumulation of Delta6-unsaturated fatty acids, this was not observed in plants transformed with N-terminally deleted forms of the desaturase. Site-directed mutagenesis was used to disrupt one of the axial heme-binding residues (histidine-41) of the Cyt b5 domain; expression of this mutant form of the Delta6-desaturase in transgenic plants failed to produce Delta6-unsaturated fatty acids. These data indicate that the Cyt b5 domain of the borage Delta6-fatty acid desaturase is essential for enzymatic activity. (+info
Structure and characterization of Ectothiorhodospira vacuolata cytochrome b(558), a prokaryotic homologue of cytochrome b(5).
A soluble cytochrome b(558) from the purple phototropic bacterium Ectothiorhodospira vacuolata was completely sequenced by a combination of automated Edman degradation and mass spectrometry. The protein, with a measured mass of 10,094.7 Da, contains 90 residues and binds a single protoheme. Unexpectedly, the sequence shows homology to eukaryotic cytochromes b(5). As no prokaryotic homologue had been reported so far, we developed a protocol for the expression, purification, and crystallization of recombinant cytochrome b(558). The structure was solved by molecular replacement to a resolution of 1.65 A. It shows that cytochrome b(558) is indeed the first bacterial cytochrome b(5) to be characterized and differs from its eukaryotic counterparts by the presence of a disulfide bridge and a four-residue insertion in front of the sixth ligand (histidine). Eukaryotes contain a variety of b(5) homologues, including soluble and membrane-bound multifunctional proteins as well as multidomain enzymes such as sulfite oxidase, fatty-acid desaturase, nitrate reductase, and lactate dehydrogenase. A search of the Mycobacterium tuberculosis genome showed that a previously unidentified gene encodes a fatty-acid desaturase with an N-terminal b(5) domain. Thus, it may provide another example of a bacterial b(5) homologue. (+info
Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells.
Cytochrome b-type NAD(P)H oxidoreductases are involved in many physiological processes, including iron uptake in yeast, the respiratory burst, and perhaps oxygen sensing in mammals. We have identified a cytosolic cytochrome b-type NAD(P)H oxidoreductase in mammals, a flavohemoprotein (b5+b5R) containing cytochrome b5 (b5) and b5 reductase (b5R) domains. A genetic approach, using BLAST searches against DBEST for FAD-, NAD(P)H-binding sequences followed by reverse transcription-PCR, was used to clone the complete cDNA sequence of human b5+b5R from the hepatoma cell line Hep 3B. Compared with the classical single-domain b5 and b5R proteins localized on endoplasmic reticulum membrane, b5+b5R also has binding motifs for heme, FAD, and NAD(P)H prosthetic groups but no membrane anchor. The human b5+b5R transcript was expressed at similar levels in all tissues and cell lines that were tested. The two functional domains b5* and b5R* are linked by an approximately 100-aa-long hinge bearing no sequence homology to any known proteins. When human b5+b5R was expressed as c-myc adduct in COS-7 cells, confocal microscopy revealed a cytosolic localization at the perinuclear space. The recombinant b5+b5R protein can be reduced by NAD(P)H, generating spectrum typical of reduced cytochrome b with alpha, beta, and Soret peaks at 557, 527, and 425 nm, respectively. Human b5+b5R flavohemoprotein is a NAD(P)H oxidoreductase, demonstrated by superoxide production in the presence of air and excess NAD(P)H and by cytochrome c reduction in vitro. The properties of this protein make it a plausible candidate oxygen sensor. (+info