Automated multiple analysis of protein structures: Application to homology modeling of cytochromes P450

Jean, P.; Pothier, Joël; Dansette, Patrick M.; Mansuy, Daniel; Viari, Alain

doi:10.1002/(sici)1097-0134(199707)28:3<388::aid-prot9>3.0.co;2-8

Cited by 21 publications

(26 citation statements)

References 67 publications

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“…Also, it does not reliably align with the sequences of known structures in areas other than the most conserved regions common to all P450 enzymes. A CYP73A1 homology model was built using the computational strategy, previously described by Jean et al [21] and further improved by Minoletti [22], that identifies substructures, or structurally conserved blocks, in the crystal structures of related proteins, and then locates similar blocks in the target sequence. Common structural blocks of the four P450 structures (P450 BM3 , P450 CAM , P450 TERP , and P450 eryF ) available from the Brookhaven PDB at the start of this work were located in the CYP73A1 sequence as represented in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The computational homology modelling strategy described by Jean et al [21] allows a reasonable prediction of the most conserved P450 substructures, although hypervariable regions cannot be predicted. Our present model was based on four crystallized bacterial enzymes (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Common structural blocks were determined previously using the GOK interactive program [21]. Side chain atoms determination, distance and dihedral constraints calculation, rotamer selection, and data analysis were performed by writing macros in BCL language from Accelrys (MSI), in AWK language, in UNIX macros, and by using the functionalities of INSIGHTII and BIOPOLYMER modules from Accelrys.…”

Section: Modelling Programs and Calculationsmentioning

confidence: 99%

“…Common structural blocks were determined for the four structures by Jean et al [21] using the program GOK and the related strategy. For specified tolerance parameters, this program performs a multiple structure comparison from internal coordinates (we used Alpha, Tau).…”

Section: Construction Of the Modelsmentioning

confidence: 99%

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Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1

Schoch

Attias

Ret

et al. 2003

European Journal of Biochemistry

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CYP73 enzymes are highly conserved cytochromes P450 in plant species that catalyse the regiospecific 4-hydroxylation of cinnamic acid to form precursors of lignin and many other phenolic compounds. A CYP73A1 homology model based on P450 experimentally solved structures was used to identify active site residues likely to govern substrate binding and regio-specific catalysis. The functional significance of these residues was assessed using site-directed mutagenesis. Active site modelling predicted that N302 and I371 form a hydrogen bond and hydrophobic contacts with the anionic site or aromatic ring of the substrate. Modification of these residues led to a drastic decrease in substrate binding and metabolism without major perturbation of protein structure. Changes to residue K484, which is located too far in the active site model to form a direct contact with cinnamic acid in the oxidized enzyme, did not influence initial substrate binding. However, the K484M substitution led to a 50% loss in catalytic activity. K484 may affect positioning of the substrate in the reduced enzyme during the catalytic cycle, or product release. Catalytic analysis of the mutants with structural analogues of cinnamic acid, in particular indole-2-carboxylic acid that can be hydroxylated with different regioselectivities, supports the involvement of N302, I371 and K484 in substrate docking and orientation.Keywords: active site; cinnamate 4-hydroxylase; homology modeling; plant cytochrome P450; site-directed mutagenesis.CYP73 designates a family of plant cytochromes P450 that evolved with or before the evolution of vascular plants. Up to 20% of the woody plant biomass is processed by CYP73 enzymes to form lignin monomers, UV-shielding or insect attracting pigments, and defensive compounds [1,2]. CYP73 enzymes belong to the same subfamily, i.e. share more than 55% amino acid identity, and catalyse the regiospecific 4-hydroxylation of trans-cinnamic acid into p-coumaric acid [3][4][5]. The importance of this reaction in plant biology seems to have precluded further evolution and diversification of the CYP73A P450 subfamily to the processing of other endogenous metabolites. CYP73A1 was one of the first plant P450 genes isolated [6]. Expression in yeast indicated that the cinnamate 4-hydroxylase (C4H) activity proceeds with a perfect coupling of oxygen consumption and reducing equivalents to produce hydroxylated substrates [3]. CYP73A1 provides a good model for determining the residues that control catalytic efficiency and optimal substrate positioning in a typical plant P450 enzyme contributing to a high throughput anabolic pathway.CYP73A1 is one of the most extensively studied plant P450 enzymes. It has a quite high substrate specificity but can accommodate a diverse array of compounds, as far as they are structural analogues of the natural substrate. Structural requirements for such analogues include a planar, aromatic structure, a small size of about two adjacent aromatic rings, and an anionic site opposite (i.e. at about 8.5 Å ) to the p...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Modelling Programs and Calculationsmentioning

confidence: 99%

Section: Construction Of the Modelsmentioning

confidence: 99%

See 2 more Smart Citations

Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1

Schoch

Attias

Ret

et al. 2003

European Journal of Biochemistry

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“…After revelation by immunoblotting of these truncated CYP2C9 with a number of anti-LKM2 sera from different patients it became clear that the epitope located in the C-terminal part of the protein was conformational (49). Deletion mutants were tested and those recognized, incorporated in a computer model of CYP2C9 (50), showed that this epitope was located close to the cytosolic surface of the protein. This epitope correspond to a cluster of three helices: helix J (314-322), K (345-356) and L (445-455).…”

Section: Epitope Mapping Of Anti·lkm2mentioning

confidence: 99%

Drug-induced immunotoxicity

Dansette

Bonierbale

Minoletti

et al. 1998

Eur. J. Drug Metab. Pharmacokinet.

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Immune-related drug responses are one of the most common sources of idiosyncratic toxicity. A number of organs may be the target of such reactions; however, this review concentrates mostly on the liver. Drug-induced hepatitis is generally divided into two categories: acute hepatitis in which the drug or a metabolite destroys a vital target in the cell; immunoallergic hepatitis in which the drug triggers an adverse immune response directed against the liver. Their clinical features are: a) low frequency; b) dose independence; c) typical immune system manifestations such as fever, eosinophilia; d) delay between the initiation of treatment and onset of the disease; e) a shortened delay upon rechallenge; and f) occasional presence of autoantibodies in the serum of patients. Such signs have been found in cases of hepatitis triggered by drugs such as halothane, tienilic acid, dihydralazine and anticonvulsants. They will be taken as examples to demonstrate the recent progress made in determining the mechanisms responsible for the disease. The following mechanisms have been postulated: 1) the drug is first metabolized into a reactive metabolite which binds to the enzyme that generated it; 2) this produces a neoantigen which, once presented to the immune system, might trigger an immune response characterized by 3) the production of antibodies recognizing both the native and/or the modified protein; 4) rechallenge leads to increased neoantigen production, a situation in which the presence of antibodies may induce cytolysis. Toxicity is related to the nature and amount of neoantigen and also to other factors such as the individual immune system. An effort should be made to better understand the precise mechanisms underlying this kind of disease and thereby identify the drugs at risk; and also the neoantigen processes necessary for their introduction into the immune system. An animal model would be useful in this regard.

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CytochromeP450

2004

Handbook of Metalloproteins

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Automated multiple analysis of protein structures: Application to homology modeling of cytochromes P450

Cited by 21 publications

References 67 publications

Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1

Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1

Drug-induced immunotoxicity

CytochromeP450

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