The 1.92-Å Structure of Streptomyces coelicolor A3(2) CYP154C1

Podust, L.M.; Kim, Young Chang; Arase, Miharu; Neely, Benjamin A.; Beck, Brian J.; Bach, Horacio; Sherman, David H.; Lamb, David C.; Kelly, Steven L.; Waterman, Michael R.

doi:10.1074/jbc.m212210200

Cited by 79 publications

(43 citation statements)

References 62 publications

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“…This structure revealed that the protein can adopt a wide open conformation, allowing direct transfer of substrates from the protein exterior to the active site. This open cleft was grossly similar to openings observed in a few recent bacterial P450 structures (4,5) and was stabilized by dimerization of the protein, which occurs both in the crystals and in solution. In the symmetric homodimer, a portion of one molecule (helices FЈ-GЈ) partially fills the cleft of the second molecule, with His-226 forming an intermolecular coordinate bond to the sixth position of the heme iron.…”

supporting

confidence: 81%

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Structure of Mammalian Cytochrome P450 2B4 Complexed with 4-(4-Chlorophenyl)imidazole at 1.9-Å Resolution

Scott

White

et al. 2004

Journal of Biological Chemistry

280

194

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A 1.9-Å molecular structure of the microsomal cytochrome P450 2B4 with the specific inhibitor 4-(4-chlorophenyl)imidazole (CPI) in the active site was determined by x-ray crystallography. In contrast to the previous experimentally determined 2B4 structure, this complex adopted a closed conformation similar to that observed for the mammalian 2C enzymes. The differences between the open and closed structures of 2B4 were primarily limited to the lid domain of helices F through G, helices B and C, the N terminus of helix I, and the ␤ 4 region. These large-scale conformational changes were generally due to the relocation of conserved structural elements toward each other with remarkably little remodeling at the secondary structure level. For example, the F and G helices were maintained with a sharp turn between them but are placed to form the exterior ceiling of the active site in the CPI complex. CPI was closely surrounded by residues from substrate recognition sites 1, 4, 5, and 6 to form a small, isolated hydrophobic cavity. The switch from open to closed conformation dramatically relocated helix C to a more proximal position. As a result, heme binding interactions were altered, and the putative NADPH-cytochrome P450 reductase binding site was reformed. This suggests a structural mechanism whereby ligand-induced conformational changes may coordinate catalytic activity. Comparison of the 2B4/CPI complex with the open 2B4 structure yields insights into the dynamics involved in substrate access, tight inhibitor binding, and coordination of substrate and redox partner binding. Cytochromes P450 (P450)1 are involved in steroidogenesis, fatty acid metabolism, synthesis of bile and retinoid acids, and production of plant toxins, but it is their function in the elimination of xenobiotics that has received the most attention. In mammals, xenobiotic metabolizing P450s play the central role in detoxification of hydrophobic drugs, carcinogens, and toxins by decreasing the lipid solubility of these chemicals and, thus, promoting excretion. In contrast to the strict substrate selectivity of classical enzymes, xenobiotic-metabolizing P450s can each bind and oxidize a set of substrates with distinct sizes, shapes, and stereochemical features. Although the variety of substrates binding to a given P450 is often broad, the oxidation of each is usually remarkably regiospecific and stereospecific.Identification of the structural basis for the specific monooxygenation and binding of a diverse but select set of substrates has been a particularly challenging goal, which is an important prerequisite for understanding selective substrate oxidation. Although the diversity of substrates might suggest an easily accessible active site, initial structures of both soluble bacterial (1) and microsomal mammalian (2) P450s revealed active sites buried within the globular structure of the protein.A few recent bacterial structures, however, suggest a "lid" domain composed of helices F and G, the motion of which controls substrate entry (3-5). Protein ...

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confidence: 81%

“…A few recent bacterial structures, however, suggest a "lid" domain composed of helices F and G, the motion of which controls substrate entry (3)(4)(5). Protein flexibility has also been implicated in dictating the regiospecificity of oxidation once a substrate is present in the active site.…”

mentioning

confidence: 99%

Structure of Mammalian Cytochrome P450 2B4 Complexed with 4-(4-Chlorophenyl)imidazole at 1.9-Å Resolution

Scott

White

et al. 2004

Journal of Biological Chemistry

280

194

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“…Together with the F helices, they constitute two layers of anti-parallel ␣-helices crossing each other at an angle of ϳ60°. A similar dimerization pattern, although with a smaller (600 Å 2 , 3.5% of the monomer surface) dimerization interface, is observed in ligand-free CYP154C1 (45) (Fig. 5B).…”

Section: Protein-protein Interactions and Bindingsupporting

confidence: 68%

“…Two potential sources of CYP130 binding cooperativity can be considered as follows: (i) multiple site cooperativity, in which two or more ligands bind simultaneously to the same protein molecule, and (ii) multimer cooperativity, where protein-protein interactions are pro- (45). Stoichiometry of CYP130 Inhibitor Binding-To address the possibility that binding cooperativity may arise from the binding of multiple inhibitor molecules in the CYP130 active site, the stoichiometry for the CYP130-econazole and CYP130-miconazole complexes was determined using the Job's titration method (22), which is based on mixing of the reactants in such a way that their molar ratio varies, whereas the total molar concentration remains constant.…”

Section: Expression and Purification Of Cyp130-cyp130mentioning

confidence: 99%

Mycobacterium tuberculosis CYP130

Ouellet

Podust

Montellano

2008

Journal of Biological Chemistry

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CYP130 is one of the 20 Mycobacterium tuberculosis cytochrome P450 enzymes, only two of which, CYP51 and CYP121, have so far been studied as individually expressed proteins. Here we characterize a third heterologously expressed M. tuberculosis cytochrome P450, CYP130, by UV-visible spectroscopy, isothermal titration calorimetry, and x-ray crystallography, including determination of the crystal structures of ligand-free and econazole-bound CYP130 at a resolution of 1.46 and 3.0 Å , respectively. Ligand-free CYP130 crystallizes in an "open" conformation as a monomer, whereas the econazole-bound form crystallizes in a "closed" conformation as a dimer. Conformational changes enabling the "open-closed" transition involve repositioning of the BC-loop and the F and G helices that envelop the inhibitor in the binding site and reshape the protein surface. Crystal structure analysis shows that the portion of the BC-loop relocates as much as 18 Å between the open and closed conformations. Binding of econazole to CYP130 involves a conformational change and is mediated by both a set of hydrophobic interactions with amino acid residues in the active site and coordination of the heme iron. CYP130 also binds miconazole with virtually the same binding affinity as econazole and clotrimazole and ketoconazole with somewhat lower affinities, which makes it a plausible target for this class of therapeutic drugs. Overall, binding of the azole inhibitors is a sequential two-step, entropydriven endothermic process. Binding of econazole and clotrimazole exhibits positive cooperativity that may reflect a propensity of CYP130 to associate into a dimeric structure.The pathogenic bacterium Mycobacterium tuberculosis continues to be an enormous threat to human health. It is responsible for more deaths worldwide than any other infectious agent, and it is the major cause of death for human immunodeficiency virus-infected individuals in developing countries. An aggravating factor associated with the global resurgence of tuberculosis is the proliferation of strains resistant to isoniazid and rifampicin, the two major frontline antitubercular drugs. Therefore, new drug strategies are needed to combat the rising incidence of tuberculosis, especially the multidrug-resistant forms, and to shorten the duration of tuberculosis treatment (1).It has been demonstrated that azole drugs such as econazole and clotrimazole, which inhibit the sterol 14␣-demethylase CYP51 and were originally developed as fungal antibiotics (2), display inhibitory potential against the latent and multidrugresistant forms of tuberculosis both in vitro and in tuberculosisinfected mice (3-7). Furthermore, econazole exhibits synergistic activities with rifampicin and isoniazid against the multidrug-resistant M. tuberculosis strains (3). The 4.4-Mb M. tuberculosis genome encodes 20 different cyp genes (8), whose biological roles are not yet understood. To date, physiological roles have been proposed for CYP125 and CYP142 in cholesterol catabolism (9) and for CYP132 in fatty acid meta...

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“…The structure of two CYP154 enzymes from Streptomyces coelicolor A3(2), CYP154C1 and CYP154A1, has already been elucidated by X-ray crystallography (18,19). CYP154C1 has been shown to convert the 12-and 14-membered ring macrolides, narbomycin and YC-17, into pikromycin and neomethymycin, respectively, in vitro (18); however, not enough substrate screening of CYP154C1 has been performed.…”

mentioning

confidence: 99%

Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces griseus Cytochrome P450 CYP154C3

Makino

Katsuyama

Otomatsu³

et al. 2014

Appl Environ Microbiol

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c Cytochrome P450 monooxygenases (P450s), which constitute a superfamily of heme-containing proteins, catalyze the direct oxidation of a variety of compounds in a regio-and stereospecific manner; therefore, they are promising catalysts for use in the oxyfunctionalization of chemicals. In the course of our comprehensive substrate screening for all 27 putative P450s encoded by the Streptomyces griseus genome, we found that Escherichia coli cells producing an S. griseus P450 (CYP154C3), which was fused C terminally with the P450 reductase domain (RED) of a self-sufficient P450 from Rhodococcus sp., could transform various steroids (testosterone, progesterone, ⌬ 4 -androstene-3,17-dione, adrenosterone, 1,4-androstadiene-3,17-dione, dehydroepiandrosterone, 4-pregnane-3,11,20-trione, and deoxycorticosterone) into their 16␣-hydroxy derivatives as determined by nuclear magnetic resonance and high-resolution mass spectrometry analyses. The purified CYP154C3, which was not fused with RED, also catalyzed the regio-and stereospecific hydroxylation of these steroids at the same position with the aid of ferredoxin and ferredoxin reductase from spinach. The apparent equilibrium dissociation constant (K d ) values of the binding between CYP154C3 and these steroids were less than 8 M as determined by the heme spectral change, indicating that CYP154C3 strongly binds to these steroids. Furthermore, kinetic parameters of the CYP154C3-catalyzed hydroxylation of ⌬ 4 -androstene-3,17-dione were determined (K m , 31.9 ؎ 9.1 M; k cat , 181 ؎ 4.5 s ؊1 ). We concluded that CYP154C3 is a steroid D-ring 16␣-specific hydroxylase which has considerable potential for industrial applications. This is the first detailed enzymatic characterization of a P450 enzyme that has a steroid D-ring 16␣-specific hydroxylation activity. C ytochrome P450 monooxygenases (P450s or CYPs) are found in all three domains of life, i.e., archaea, bacteria, and eukarya, and are classified into more than 1,000 families by their amino acid sequence homology (1). They have the ability to hydroxylate inactive carbons of hydrocarbons or aromatic compounds regioand stereospecifically (2, 3). In animals, P450s are mainly involved in xenobiotic metabolism and steroid biosynthesis (4, 5). In plants, many P450s are involved in the biosynthesis of plant hormones and secondary metabolites (6). In contrast, the functions of bacterial P450s are largely unknown, although some bacterial P450s are required for secondary metabolite biosynthesis and assimilation of terpenoids, such as cineol or terpineol (7-10). In spite of their unknown physiological functions, however, bacterial P450s have attracted much attention as a rich resource for new biocatalysts for use in the oxyfunctionalization of chemicals (11, 12). For example, microbial conversion of steroid precursors is widely used for large-scale synthesis of steroid drugs (13-17).CYP154 family members are widely found in actinomycetes, and many putative CYP154 genes have been found in the genomes of Streptomyces species. The structu...

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The 1.92-Å Structure of Streptomyces coelicolor A3(2) CYP154C1

Cited by 79 publications

References 62 publications

Structure of Mammalian Cytochrome P450 2B4 Complexed with 4-(4-Chlorophenyl)imidazole at 1.9-Å Resolution

Structure of Mammalian Cytochrome P450 2B4 Complexed with 4-(4-Chlorophenyl)imidazole at 1.9-Å Resolution

Mycobacterium tuberculosis CYP130

Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces griseus Cytochrome P450 CYP154C3

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