Abstract:
:The structural determinants of substrate specificity of human liver cytochrome P450 2C8 (CYP2C8) were investigated using site-directed mutants chosen on the basis of a preliminary substrate pharmacophore and a three-dimensional (3D) model. Analysis of the structural features common to CYP2C8 substrates exhibiting a micromolar K(m) led to a substrate pharmacophore in which the site of oxidation by CYP2C8 is 12.9, 8.6, 4.4, and 3.9 A from features that could establish ionic or hydrogen bonds, and hydrophobic interactions with protein amino acid residues. Comparison of this pharmacophore with a 3D model of CYP2C8 constructed using the X-ray structure of CYP2C5 suggested potential CYP2C8 amino acid residues that could be involved in substrate recognition. Twenty CYP2C8 site-directed mutants were constructed and expressed in yeast to compare their catalytic activities using five CYP2C8 substrates that exhibit different structures and sizes [paclitaxel, fluvastatin, retinoic acid, a sulfaphenazole derivative (DMZ), and diclofenac]. Mutation of arginine 241 had marked effects on the hydroxylation of anionic substrates of CYP2C8 such as retinoic acid and fluvastatin. Serine 100 appears to be involved in hydrogen bonding interactions with a polar site of the CYP2C8 substrate pharmacophore, as shown by the 3-4-fold increase in the K(m) of paclitaxel and DMZ hydroxylation after the S100A mutation. Residues 114, 201, and 205 are predicted to be in close contact with substrates, and their mutations lead either to favorable hydrophobic interactions or to steric clashes with substrates. For instance, the S114F mutant was unable to catalyze the 6alpha-hydroxylation of paclitaxel. The S114F and F205A mutants were the best catalysts for retinoic acid and paclitaxel (or fluvastatin) hydroxylation, respectively, with k(cat)/K(m) values 5 and 2.1 (or 2.4) times higher, respectively, than those found for CYP2C8. Preliminary experiments of docking of the substrate into the experimentally determined X-ray structure of substrate-free CYP2C8, which became available quite recently [Schoch, G. A., et al. (2004) J. Biol. Chem. 279, 9497], were consistent with key roles for S100, S114, and F205 residues in substrate binding. The results suggest that the effects of mutation of arginine 241 on anionic substrate hydroxylation could be indirect and result from alterations of the packing of helix G with helix B'.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Melet A,Marques-Soares C,Schoch GA,Macherey AC,Jaouen M,Dansette PM,Sari MA,Johnson EF,Mansuy Ddoi
10.1021/bi0489309subject
Has Abstractpub_date
2004-12-14 00:00:00pages
15379-92issue
49eissn
0006-2960issn
1520-4995journal_volume
43pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::The effects of ions on the interaction of lac repressor protein and operator DNA have been studied by the membrane filter technique. The equilibrium association constant was determined as a function of monovalent and divalent cation concentrations, anions, and pH. The binding of repressor and operator is extremely sen...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00516a027
更新日期:1981-06-23 00:00:00
abstract::The physical origin of the extremely high thermal stability of tetraether liposomes composed of the polar lipid fraction E (PLFE) from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius has been investigated. The leakage rate of trapped 5, 6-carboxyfluorescein (5(6)CF) and the proton permeability in PLFE ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi972163e
更新日期:1998-01-06 00:00:00
abstract::The addition of 10(-9) M insulin to a suspension of rat liver plasma membranes increases the overall lipid microviscosity, eta, by about 10--20%. The effect is confined to physiological concentrations of the hormone and is highly specific. The specificity was demonstrated in experiments where insulin analogues were ad...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00570a009
更新日期:1979-02-06 00:00:00
abstract::The pyridoxal 5'-phosphate (PLP)-dependent cystathionine beta-lyase (CBL) was previously found to be inhibited by the natural toxins rhizobitoxine and l-aminoethoxyvinylglycine (AVG). The present study characterizes the interaction of Escherichia coli CBL with AVG and methoxyvinylglycine (MVG) by a combination of kine...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi970630m
更新日期:1997-10-14 00:00:00
abstract::Electron spin polarized electron paramagentic resonance (ESP EPR) spectra were obtained with deuterated iron-removed photosynthetic bacterial reaction centers (RCs) to specifically investigate the effect of the rate of primary charge separation, metal-site occupancy, and H-subunit content on the observed P865+QA- char...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9630319
更新日期:1997-07-15 00:00:00
abstract::The melibiose permease of Salmonella typhimurium (MelB(St)) catalyzes symport of melibiose with Na(+), Li(+), or H(+), and bioinformatics analysis indicates that a conserved Gly117 (helix IV) is part of the Na(+)-binding site. We mutated Gly117 to Ala, Pro, Trp, or Arg; the effects on melibiose transport and binding o...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi300230h
更新日期:2012-04-03 00:00:00
abstract::A small metal-binding protein (SmbP) with no known similarity to other proteins in current databases was isolated and characterized from the periplasm of Nitrosomonas europaea. The primary structure of this small (9.9 kDa) monomeric protein is characterized by a series of 10 repeats of a seven amino acid motif and an ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi049318k
更新日期:2004-09-07 00:00:00
abstract::One-dimensional 1H NMR was employed to study the effects of Ca2+ and Lu3+ binding on the apo and calcium-saturated forms of dog bone Gla protein (BGP, osteocalcin). Titration of apo dog BGP with Ca2+ in 20 mM NaCl showed spectral perturbations consistent with the binding of 5 mol equiv of calcium in the NMR slow-excha...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00093a012
更新日期:1993-10-26 00:00:00
abstract::HTC cells have been labeled by short exposures to [3H]thymidine in order to identify newly synthesized DNA. By either isolating nuclei directly or isolating them after an extensive fixation with formaldehyde, we have been able to identify two phases in the maturation process of newly replicated chromatin. The first ph...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00302a036
更新日期:1984-03-27 00:00:00
abstract::Two hybrid ribulose-1,5 bisphosphate carboxylase/oxygenase (RubisCO) enzymes were constructed using RubisCO small subunit genes (rbcS) from two eucaryotic marine organisms, Cylindrotheca sp. N1 and Olisthodiscus luteus, cloned downstream of the RubisCO large subunit gene (rbcL) of the cyanobacterium Synechococcus PCC ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00139a018
更新日期:1992-06-23 00:00:00
abstract::Exoenzyme S (ExoS) is a bifunctional virulence factor directly translocated into eukaryotic cells by the type III secretory process of Pseudomonas aeruginosa. Bacterial translocation of ExoS into epithelial cells is associated with diverse effects on cell function, including inhibition of growth, alterations in cell m...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi025826n
更新日期:2002-07-30 00:00:00
abstract::Activated receptors with a tyrosine kinase activity induce a variety of responses like changes in the differentiation and mitogenic status of cells. These responses are mediated in part by p21ras. Some of these activated receptors induce in certain cell types a pronounced, but transient, increase in Ras-GTP. We have s...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00189a053
更新日期:1994-06-14 00:00:00
abstract::Here we explore the use of ethidium to determine relative affinities of different gene delivery vectors for DNA and describe an improved method for studying the interaction. Specifically, we investigate the binding of poly(amidoamine) dendrimers and show that the DNA-dendrimer-ethidium system is far from thermodynamic...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi1015887
更新日期:2011-02-22 00:00:00
abstract::The functional interaction of the recombinant alpha 2 adrenergic receptor subtypes, alpha 2-C10 (the human platelet alpha 2 receptor, equivalent to the alpha 2 A subtype) and alpha 2-C4 (an alpha 2 receptor subtype cloned from a human kidney cDNA library), with G proteins was characterized in an in vitro reconstitutio...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00227a024
更新日期:1991-04-02 00:00:00
abstract::The disulfide bond location of a homodimeric peptide, prothoracicotropic hormone (PTTH) of the silkworm, Bombyx mori, was determined by a combination of partial reduction and sequence analysis of peptide fragments generated through a partial reduction of PTTH followed by alkylation and enzyme digestion. The partial re...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00185a031
更新日期:1994-05-17 00:00:00
abstract::Anastellin is a small recombinant fragment derived from the extracellular matrix protein fibronectin; it comprises the first type III (FN3) domain without the two N-terminal β-strands. It inhibits angiogenesis, tumor growth, and metastasis in mouse models and requires endogenous fibronectin for its in vivo anti-angiog...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.7b00633
更新日期:2017-09-05 00:00:00
abstract::The far-ultraviolet circular dichroism spectra of bovine and human prothrombin, prothrombin fragment 1, prethrombin 1, prothrombin fragment 2, and prethrombin 2 (prethrombin 2des(1--13)) were determined and the method of Chen et al. [Chen, Y. H., Yang, J. T., & Martinez, H. M. (1972) Biochemistry 11, 4120--4131; Chen,...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00577a017
更新日期:1979-05-15 00:00:00
abstract::The duplex genome of Escherichia coli virus M13mp10 was modified at a unique site to contain N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG8-ABP), the major carcinogen-DNA adduct of the human bladder carcinogen 4-aminobiphenyl. A tetradeoxynucleotide containing a single dG8-ABP residue was synthesized by reacting 5'-d(TpG...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00385a019
更新日期:1987-06-02 00:00:00
abstract::Chemical deglycosylation of gonadotropins with hydrogen fluoride (HF) has facilitated the investigation of the structure-function relationship of the individual peptide and oligosaccharide moieties in the mechanism of hormone action. These studies have dealt almost exclusively with lutropin or human choriogonadotropin...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00361a030
更新日期:1986-07-01 00:00:00
abstract::Three activities on the pathway of purine biosynthesis de novo in chicken liver, namely, glycinamide ribonucleotide synthetase, glycinamide ribonucleotide transformylase, and aminoimidazole ribonucleotide synthetase, have been found to reside on the same polypeptide chain. Three diverse purification schemes, utilizing...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00346a006
更新日期:1985-12-03 00:00:00
abstract::Nucleotide-induced conformational changes of the catalytic beta subunits play a crucial role in the rotary mechanism of F(1)-ATPase. To gain insights into the energetic bases that govern the recognition of nucleotides by the isolated beta subunit from thermophilic Bacillus PS3 (Tbeta), the binding of this monomer to M...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi1006767
更新日期:2010-06-29 00:00:00
abstract::Dehaloperoxidase (DHP), discovered in the marine terebellid polychaete Amphitrite ornata, is the first heme-containing globin with a peroxidase activity. The sequence and crystal structure of DHP argue that it evolved from an ancient O(2) transport and storage globin. Thus, DHP retains an oxygen carrier function but a...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi2009129
更新日期:2011-09-27 00:00:00
abstract::A positional-scanning combinatorial protocol was employed to optimize the deoxyribose-based cleavage of B-form DNA by Ni(II) x Xaa-Xaa-His metallopeptides. This procedure employed 18 naturally occurring amino acids (excluding Cys and Trp) to generate two libraries in which the first and second positions of the peptide...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi982587o
更新日期:1999-02-16 00:00:00
abstract::The essential active site Fe3+ of protocatechuate 3,4-dioxygenase [3, 4-PCD, subunit structure (alphabetaFe3+)12] is bound by axial ligands, Tyr447 (147beta) and His462 (162beta), and equatorial ligands, Tyr408 (108beta), His460 (160beta), and a solvent OH- (Wat827). Recent X-ray crystallographic studies have shown th...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi972047b
更新日期:1998-02-24 00:00:00
abstract::Staphylococcus aureus is a leading cause of surgical site infections that results in increased hospital stays due to the development of chronic wounds. Little is known about factors involved in S. aureus' ability to prevent wounds from healing. We discovered a novel secreted protein produced by a surgical site isolate...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.5b00523
更新日期:2015-08-11 00:00:00
abstract::Two antisera used in the radioimmunoassay for human fibrinopeptide A (FPA) which appear to have different immunochemical specificities have been tested for cross-reactivity with fibrinogen and with three fragments of fibrinogen which contain the FPA sequence. These fragments were the three-chain, NH2-terminal disulfid...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00651a004
更新日期:1976-03-23 00:00:00
abstract::Cyanobactin heterocyclases share the same catalytic domain (YcaO) as heterocyclases/cyclodehydratases from other ribosomal peptide (RiPPs) biosynthetic pathways. These enzymes process multiple residues (Cys/Thr/Ser) within the same substrate. The processing of cysteine residues proceeds with a known order. We show the...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.9b00084
更新日期:2019-04-23 00:00:00
abstract::Nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes or ketones with the production of H(2)O(2) and nitrite. The flavoenzyme is a new member of the acyl-CoA dehydrogenase (ACAD) family, but it does not react with acyl-CoA substrates. We presen...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi051966w
更新日期:2006-01-31 00:00:00
abstract::In the Arc repressor dimer, the side chains of Ile37 and Val41 in alpha-helix B pack against each other and against the symmetry-related side chains of Ile37' and Val41' in alpha-helix B' to form part of the hydrophobic core and the dimer interface. Following combinatorial mutagenesis of these positions, only the wild...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00010a025
更新日期:1995-03-14 00:00:00
abstract::Methenyltetrahydromethanopterin (methenyl-H(4)MPT(+)) cyclohydrolase (Mch) catalyzes the interconversion of methenyl-H(4)MPT(+) and formyl-H(4)MPT in the one-carbon energy metabolism of methanogenic, methanotrophic, and sulfate-reducing archaea and of methylotrophic bacteria. To understand the catalytic mechanism of t...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi300777k
更新日期:2012-10-23 00:00:00