Crystal Structure of Cytochrome P450cam Complexed with Its Catalytic Product, 5-exo-Hydroxycamphor

Li, Huiying; Narasimhulu, Shakunthala; Havran, Lisa M.; Winkler, Jeffery; Poulos, T.L.

doi:10.1021/ja00128a019

Cited by 69 publications

(60 citation statements)

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“…Close proximity to the heme iron was suggested for 22-HC earlier based on NMR studies (14). Crystallographic observation of the hydroxyl-heme iron interaction was made with the hydroxylated product of P450cam (15,16). Upon 22-HC binding, the conformation of the Thr291 side chain is slightly changed so the interaction between the carbonyl oxygen of Gly287 and the hydroxyl group of Thr291 is loosened, which allows a new water molecule to bind in the I-helix groove (Fig.…”

Section: Resultsmentioning

confidence: 94%

Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system

Strushkevich

Mackenzie

Cherkesova

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

290

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In humans, the precursor to all steroid hormones, pregnenolone, is synthesized from cholesterol by an enzyme complex comprising adrenodoxin reductase (AdR), adrenodoxin (Adx), and a cytochrome P450 (P450scc or CYP11A1). This complex not only plays a key role in steroidogenesis, but also has long been a model to study electron transfer, multistep catalysis, and C–C bond cleavage performed by monooxygenases. Detailed mechanistic understanding of these processes has been hindered by a lack of structural information. Here we present the crystal structure of the complex of human Adx and CYP11A1—the first of a complex between a eukaryotic CYP and its redox partner. The structures with substrate and a series of reaction intermediates allow us to define the mechanism underlying sequential hydroxylations of the cholesterol and suggest the mechanism of C–C bond cleavage. In the complex the [2Fe-2S] cluster of Adx is positioned 17.4 Å away from the heme iron of CYP11A1. This structure suggests that after an initial protein–protein association driven by electrostatic forces, the complex adopts an optimized geometry between the redox centers. Conservation of the interaction interface suggests that this mechanism is common for all mitochondrial P450s.

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Section: Resultsmentioning

confidence: 94%

Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system

Strushkevich

Mackenzie

Cherkesova

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

290

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“…The structure of Vdh-K1 with bound 25(OH)VD 3 revealed that the 3␤-OH group of 25(OH)VD 3 is a sixth coordination ligand that is positioned very close to the heme iron. Ligation of the OH group to the heme iron was previously reported in the structure of P450 cam complexed with the product 5-exo-hydroxycamphor (42). In addition, several mammalian CYPs show the reverse type I spectral change in response to the addition of a substrate or inhibitor carrying OH groups (Ref.…”

Section: Discussionmentioning

confidence: 85%

Structural Evidence for Enhancement of Sequential Vitamin D3 Hydroxylation Activities by Directed Evolution of Cytochrome P450 Vitamin D3 Hydroxylase

Yasutake

Fujii

Nishioka

et al. 2010

Journal of Biological Chemistry

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Vitamin D 3 hydroxylase (Vdh) isolated from actinomycete Pseudonocardia autotrophica is a cytochrome P450 (CYP) responsible for the biocatalytic conversion of vitamin D 3 (VD 3 ) to 1␣,25-dihydroxyvitamin D 3 (1␣,25(OH) 2 VD 3 ) by P. autotrophica. Although its biological function is unclear, Vdh is capable of catalyzing the two-step hydroxylation of VD 3 , i.e. the conversion of VD 3 to 25-hydroxyvitamin D 3 (25(OH)VD 3 ) and then of 25(OH)VD 3 to 1␣,25(OH) 2 VD 3 , a hormonal form of VD 3 . Here we describe the crystal structures of wild-type Vdh (Vdh-WT) in the substrate-free form and of the highly active quadruple mutant (Vdh-K1) generated by directed evolution in the substrate-free, VD 3 -bound, and 25(OH)VD 3 -bound forms. Vdh-WT exhibits an open conformation with the distal heme pocket exposed to the solvent both in the presence and absence of a substrate, whereas Vdh-K1 exhibits a closed conformation in both the substrate-free and substrate-bound forms. The results suggest that the conformational equilibrium was largely shifted toward the closed conformation by four amino acid substitutions scattered throughout the molecule. The substratebound structure of Vdh-K1 accommodates both VD 3 and 25(OH)VD 3 but in an anti-parallel orientation. The occurrence of the two secosteroid binding modes accounts for the regioselective sequential VD 3 hydroxylation activities. Moreover, these structures determined before and after directed evolution, together with biochemical and spectroscopic data, provide insights into how directed evolution has worked for significant enhancement of both the VD 3 25-hydroxylase and 25(OH)VD 3 1␣-hydroxylase activities.3 is a B-ring opening secosteroid involved in a wide variety of biological functions in mammals (1). In humans, VD 3 is converted into its physiologically active form, 1␣,25-dihydroxyvitamin D 3 (1␣,25(OH) 2 VD 3 ), via hydroxylation reactions that are catalyzed by several cytochrome P450s (CYPs) (1, 2). The first hydroxylation is done at the C25 position of VD 3 by CYP27A1 (2, 3) and CYP2R1 (2, 4) in the liver to produce 25-hydroxyvitamin D 3 (25(OH)VD 3 ). The second proceeds at the C1␣ position of 25(OH)VD 3 by CYP27B1 in the kidney (5) (Fig. 1). The final product, 1␣,25(OH) 2 VD 3 , functions as a hormone with a critical role in maintaining calcium and phosphate homeostasis as well as in controlling the differentiation and proliferation of multiple cell types (1, 2, 6). Indeed, the many symptoms associated with VD 3 deficiency and the VD metabolic disorder, which include psoriasis, osteoporosis, rickets, and hypoparathyroidism, are treated using 1␣,25(OH) 2 VD 3 and its derivatives (1).Although the chemical synthesis of 1␣,25(OH) 2 VD 3 from cholesterol is an established method, it is inefficient, the maximum yield is no more than 1% (7). Alternatively, biocatalytic conversion by the actinomycete Pseudonocardia autotrophica is currently in practical use for the industrial production of 1␣,25(OH) 2 VD 3 (8, 9). We have recently cloned the gene encoding the VD 3 hydroxy...

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“…Conformational changes associated with substrate recognition, enzyme reduction and O 2 binding may be coupled to the position of the catalytically important water and Thr-252 side chain (44). It has been shown that the product complex closely resembles the closed substratebound conformation (45), and thus product release would necessitate reopening of the substrate channel. The release mechanism and its trigger are currently not fully understood.…”

Section: Discussionmentioning

confidence: 99%

Double electron–electron resonance shows cytochrome P450cam undergoes a conformational change in solution upon binding substrate

Stoll

Lee

Zhang

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Although cytochrome P450cam from Pseudomonas putida , the archetype for all heme monooxygenases, has long been known to have a closed active site, recent reports show that the enzyme can also be crystallized in at least two clusters of open conformations. This suggests that the enzyme may undergo significant conformational changes during substrate binding and catalytic turnover. However, these conformations were observed in the crystalline state, and information is needed about the conformations that are populated in solution. In this study, double electron–electron resonance experiments were performed to observe substrate-induced changes in distance as measured by the dipolar coupling between spin labels introduced onto the surface of the enzyme on opposite sides of the substrate access channel. The double electron–electron resonance data show a decrease of 0.8 nm in the distance between spin labels placed at S48C and S190C upon binding the substrate camphor. A rotamer distribution model based on the crystal structures adequately describes the observed distance distributions. These results demonstrate conclusively that, in the physiologically relevant solution state, the substrate-free enzyme exists in the open P450cam-O conformation and that camphor binding results in conversion to the closed P450cam-C form. This approach should be useful for investigating many other P450s, including mammalian forms, in which the role of conformational change is of central importance but not well understood.

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Crystal Structure of Cytochrome P450cam Complexed with Its Catalytic Product, 5-exo-Hydroxycamphor

Cited by 69 publications

References 0 publications

Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system

Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system

Structural Evidence for Enhancement of Sequential Vitamin D3 Hydroxylation Activities by Directed Evolution of Cytochrome P450 Vitamin D3 Hydroxylase

Double electron–electron resonance shows cytochrome P450cam undergoes a conformational change in solution upon binding substrate

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