Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome b5.

Bodman, Susanne B. von; Schuler, Mary A.; Jollie, David; Sligar, Stephen G.

doi:10.1073/pnas.83.24.9443

Cited by 179 publications

(139 citation statements)

References 15 publications

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“…The expression and purification of cyt b 5 were carried out by using a method described previously (41). Purified proteins with the R/Z value (A 412 /A 280 ) greater than 5.5 were employed.…”

Section: Methodsmentioning

confidence: 99%

NMR Study on the Structural Changes of Cytochrome P450cam upon the Complex Formation with Putidaredoxin

Tosha

Shiro

Takahashi

et al. 2003

Journal of Biological Chemistry

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We investigated putidaredoxin-induced structural changes in carbonmonoxy P450cam by using NMR spectroscopy. The resonance from the ␤-proton of the axial cysteine was upfield shifted by 0.12 ppm upon the putidaredoxin binding, indicating that the axial cysteine approaches to the heme-iron by about 0.1 Å. The approach of the axial cysteine to the heme-iron would enhance the electronic donation from the axial thiolate to the heme-iron, resulting in the enhanced heterolysis of the dioxygen bond. In addition to the structural perturbation on the axial ligand, the structural changes in the substrate and ligand binding site were observed. The resonances from the 5-exo-and 9-methyl-protons of d-camphor, which were newly identified in this study, were upfield shifted by 1.28 and 0.20 ppm, respectively, implying that d-camphor moves to the heme-iron by 0.15-0.7 Å. Based on the radical rebound mechanism, the approach of d-camphor to the heme-iron could promote the oxygen transfer reaction. On the other hand, the downfield shift of the resonance from the ␥-methyl group of Thr-252 reflects the movement of the side chain away from the heme-iron by ϳ0.25 Å. Because Thr-252 regulates the heterolysis of the dioxygen bond, the positional rearrangement of Thr-252 might assist the scission of the dioxygen bond. We, therefore, conclude that putidaredoxin induces the specific heme environmental changes of P450cam, which would facilitate the oxygen activation and the oxygen transfer reaction.Cytochrome P450 (P450) 1 is a heme-containing monooxygenase, which catalyzes the hydroxylation reactions of a wide variety of natural and unnatural substrates such as steroids, fatty acids, hydrocarbons, and xenobiotics (1). The P450 catalysis reaction requires two electrons from NADH or NADPH through the redox-linked proteins. In microsomal P450s, the electron transfer (ET) from NADPH to P450s is mediated by NADPH-P450 reductase containing FMN and FAD as the redox centers. In contrast, the electron from NADH or NADPH is sequentially transferred to ferredoxin reductase, ferredoxin, and finally P450 in mitochondrial and bacterial systems. The ET reaction between P450 and its redox partner is essential for the subsequent activation of molecular oxygen and the monooxygenation reaction.To understand the mechanism of the ET reaction in P450 and its redox partner system, the ET reaction between cytochrome P450cam (P450cam) from Pseudomonas putida, one of the bacterial P450s, and its redox partner, putidaredoxin (Pdx), has intensively been investigated. P450cam catalyzes the regio-and stereo-specific hydroxylation of its substrate, d-camphor (1, 2) by accepting two electrons from NADH. The ET from NADH to P450cam is sequentially mediated by a flavin group of putidaredoxin reductase and a [2Fe-2S] center of putidaredoxin (Pdx). With the availability of the P450cam (3) and Pdx (4, 5) structures, many investigators have performed the kinetic (6 -9), mutational (10 -14), and theoretical (15, 16) studies to clarify the molecular mechanism for the ET reaction ...

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Section: Methodsmentioning

confidence: 99%

NMR Study on the Structural Changes of Cytochrome P450cam upon the Complex Formation with Putidaredoxin

Tosha

Shiro

Takahashi

et al. 2003

Journal of Biological Chemistry

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“…The DNA sequences from bovine, rabbit and human cDNA libraries have corrected several errors in the earlier aminoacid sequences (Miyata, Nagata, Yamazoe & Kato, 1989;Yoo & Steggles, 1988). In addition the cytochrome b5 corresponding to the rat and the bovine aminoacid sequences, as well as several mutant forms, have been expressed in Escherichia coli using fully synthetic genomic material (Beck von Bodman, Schuler, Jollie & Sligar, 1986;Funk et al, 1990).…”

confidence: 99%

Refinement and structural analysis of bovine cytochrome b5 at 1.5 Å Resolution

Durley¹,

Mathews²

1996

Acta Cryst D

146

198

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The structure of bovine liver cytochrome bs, a soluble 93-residue proteolytic fragment of a 16 kDa. membranebound hemoprotein, initially solved at 2.0A resolution, has been refined at 1.5/~ using data collected on a diffractometer. Refinement to 2.0/~ resolution used the Hendrickson-Konnert procedure PROLSQ and was then extended to 1.5 ~ resolution using the program PROFFT. Only residues 3-87 could be identified in the model and these residues together with 93 water molecules gave an agreement factor of R = 0.161 for data in the resolution range 1.5-5/~,. The structure was finally refined using the program X-PLOR, which enabled alternate conformers to be modelled for several surface side chains. Residues 1 and 2 at the amino terminus of the protein and residue 88 near the carboxyl terminus could be identified from these electron-density maps. However the remaining disordered carboxy-terminal residues could not successfully be included in the model. A total of 117 solvent molecules were included in the final refinement to give R =0.164 for the data between 1.5 and 10/~.

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“…In terms of molecular biological characterization of these electron transport systems, a clone of the gene coding for rat liver NADPH Cyt P-450 reductase and Cyt b5 have been obtained, but no success at cloning the complete NADH Cyt b5 reductase gene has been realized. A totally synthetic gene coding for rat liver Cyt bs has been constructed in our laboratory and this protein expressed as 10% of total Escherichia coli cell protein (22).…”

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confidence: 99%

Purification and Characterization of Microsomal Cytochrome b₅ and NADH Cytochrome b₅ Reductase from Pisum sativum

Jollie

Sligar²,

Schuler³

1987

Plant Physiol.

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In this communication we document the reproducible protocols for the purification of milligram quantities of cytochrome b5 and NADH-cytochrome b5 reductase from the microsomal fraction of Pisum sativum. The cytochrome b5 component of this NADH linked electron transport chain was found to have a molecular mass of 16,400 daltons and the reductase a molecular mass of 34,500 daltons. These components could be reconstituted into a functional NADH oxidase activity active in the reduction of exogenous cytochrome c or ferricyanide. In the latter assay the purified reductase exhibited a turnover number of 22,000 per minute. The aminoterminal amino acid sequence of the cytochrome b5 component was determined by sequential Edmund degredation, thus providing crucial information for the efficient cloning of this central protein of plant microsomal electron transfer.The microsomal electron transport systems of mammalian tissue play important roles in a variety of oxidative reactions including fatty acid desaturation and the support of mixed function oxidative biotransformations catalyzed by the Cyt P-450 monooxygenases. Reductive reactions of the latter class involve NADPH dehydrogenation by a flavoprotein, NADPH-Cyt P-450 reductase, containing both FAD and FMN prosthetic groups, followed by sequential one electron tansfers to the cytochrome P450 heme center. In addition, some P450 biotransformations can be mediated by electron transfer from a short redox transfer chain which links to the dephosphorylated form of pyridine nucleotide, NADH. This system includes NADH-Cyt b5 reductase, a flavoprotein with a single FAD prosthetic group, and the heme protein Cyt b5. Both the NADPH and NADH flavoprotein reductases as well as Cyt b5 have been purified to homogeneity from a variety of mammalian microsomal fractions and have received intensive study during the past decade (20,23,24). Both of these proteins of the NADH linked chain are amphipathic (15) in nature, consisting oftwo definite domains, a water soluble catalytic portion and a hydrophobic fragment that binds the protein to the microsomal membrane. The catalytic domain can be separated from the holenzyme by limited proteolytic digestion ofthe microsomal fraction (9, 21). Alternately, the intact enzyme can be obtained by detergent solubilization (16,20,24 Unfortunately, much less is known about the corresponding electron transport and P-450 monoxygenase systems from plant tissue. Galle et al. (5) have recently purified an NADH-ferricyanide reductase from potato tuber that is apparently involved in the desaturation of oleate to form linoleic acid in higher plants. Two mitochondrial forms of a reductase have also been isolated by Klein and Burke (8), the first of which contained two peptide fragments and was specific for NADH whereas the second was apparently comprised of five polypeptides, displayed a broad pH maxima, and utilized both NADH and NADPH. Efforts at the isolation of a Cyt P-450 reductase from the microsomal fraction of higher plants began with the partial pur...

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Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome b5.

Cited by 179 publications

References 15 publications

NMR Study on the Structural Changes of Cytochrome P450cam upon the Complex Formation with Putidaredoxin

NMR Study on the Structural Changes of Cytochrome P450cam upon the Complex Formation with Putidaredoxin

Refinement and structural analysis of bovine cytochrome b5 at 1.5 Å Resolution

Purification and Characterization of Microsomal Cytochrome b₅ and NADH Cytochrome b₅ Reductase from Pisum sativum

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Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome b5.

Cited by 179 publications

References 15 publications

NMR Study on the Structural Changes of Cytochrome P450cam upon the Complex Formation with Putidaredoxin

NMR Study on the Structural Changes of Cytochrome P450cam upon the Complex Formation with Putidaredoxin

Refinement and structural analysis of bovine cytochrome b5 at 1.5 Å Resolution

Purification and Characterization of Microsomal Cytochrome b5 and NADH Cytochrome b5 Reductase from Pisum sativum

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Purification and Characterization of Microsomal Cytochrome b₅ and NADH Cytochrome b₅ Reductase from Pisum sativum