Substrate specificity of 6-deoxyerythronolide B hydroxylase, a bacterial cytochrome P450 of erythromycin A biosynthesis

Andersen, John F.; Tatsuta, Kuniaki; Gunji, Hiroki; Ishiyama, Takashi; Hutchinson, C. Richard

doi:10.1021/bi00059a004

Cited by 85 publications

(66 citation statements)

References 33 publications

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“…Having 46% identity, the enzymes have different enzymatic activity. Specifically, EryF catalyzes C-6 hydroxylation of the 6-deoxyerythronolide B (Scheme 1) in the biosynthesis of erythromycin (47). CYP107B1 shows no detectable activity toward 6-deoxyerythronolide B (48), and its function remains unknown.…”

Section: Resultsmentioning

confidence: 99%

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

Podust

Kim

Arase

et al. 2003

Journal of Biological Chemistry

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Evolutionary links between cytochrome P450 monooxygenases, a superfamily of extraordinarily divergent heme-thiolate proteins catalyzing a wide array of NADPH/NADH-and O 2 -dependent reactions, are becoming better understood because of availability of an increasing number of fully sequenced genomes. Among other reactions, P450s catalyze the site-specific oxidation of the precursors to macrolide antibiotics in the genus Streptomyces introducing regiochemical diversity into the macrolide ring system, thereby significantly increasing antibiotic activity. Developing effective uses for Streptomyces enzymes in biosynthetic processes and bioremediation requires identification and engineering of additional monooxygenases with activities toward a diverse array of small molecules. To elucidate the molecular basis for substrate specificity of oxidative enzymes toward macrolide antibiotics, the x-ray structure of CYP154C1 from Streptomyces coelicolor A3(2) was determined (Protein Data Bank code 1GWI). Relocation of certain common P450 secondary structure elements, along with a novel structural feature involving an additional ␤-strand transforming the five-stranded ␤-sheet into a six-stranded variant, creates an open cleft-shaped substrate-binding site between the two P450 domains. High sequence similarity to macrolide monooxygenases from other microbial species translates into catalytic activity of CYP154C1 toward both 12-and 14-membered ring macrolactones in vitro.

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Section: Resultsmentioning

confidence: 99%

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

Podust

Kim

Arase

et al. 2003

Journal of Biological Chemistry

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“…1) (1)(2)(3). The genetic manipulation of macrocyclic antibiotic biosynthetic pathways, including that of erythromycin, is currently under investigation as a route for the production of novel antibiotics (4).…”

Section: P450 Eryfmentioning

confidence: 99%

“…The crystal structure of P450 eryF shows that the hydrogen bonding interactions normally satisfied by the threonine residue are replaced by hydrogen bonding interactions with the 5-hydroxyl group of the substrate (10). The resulting requirement for substrate-assisted catalysis in the 6-hydroxylation of 6-DEB makes P450 eryF an ineffective catalyst for the oxidation of alternative substrates, even of closely related 6-DEB analogues (2). Replacement of Ala 245 by a serine or threonine reportedly decreases the rate of 6-DEB hydroxylation to 10% and 1%, respectively, of the wild-type activity (10).…”

Section: P450 Eryfmentioning

confidence: 99%

“…, and A245V Mutants-P450 eryF in the original pKOS180 -125e vector was expressed in E. coli DH5␣ cells, and the protein was purified using Q-Sepharose, Mono Q, and Superose 6 chromatographies in a yield of ϳ1 mg/liter of culture (2). In order to improve the expression level and to simplify the purification, we put a sequence coding for a 6-His tag at the N terminus of the gene and subcloned the gene into the pCWori ϩ vector.…”

Section: Expression and Characterization Of The P450 Eryf A245t A245smentioning

confidence: 99%

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An A245T Mutation Conveys on Cytochrome P450eryF the Ability to Oxidize Alternative Substrates

Hong

Tschirret-Guth

Montellano

2000

Journal of Biological Chemistry

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Cytochrome P450 eryF (CYP107A1), which hydroxylates deoxyerythronolide B in erythromycin biosynthesis, lacks the otherwise highly conserved threonine that is thought to promote O-O bond scission. The role of this threonine is satisfied in P450 eryF by a substrate hydroxyl group, making deoxyerythronolide B the only acceptable substrate. As shown here, replacement of Ala 245 by a threonine enables the oxidation of alternative substrates using either H 2 O 2 or O 2 /spinach ferredoxin/ferredoxin reductase as the source of oxidizing equivalents. Testosterone is oxidized to 1-, 11␣-, 12-, and 16␣-hydroxytestosterone. A kinetic solvent isotope effect of 2.2 indicates that the A245T mutation facilitates dioxygen bond cleavage. This gain-of-function evidence confirms the role of the conserved threonine in P450 catalysis. Furthermore, a Hill coefficient of 1.3 and dependence of the product distribution on the testosterone concentration suggest that two testosterone molecules bind in the active site, in accord with a published structure of the P450 eryF -androstenedione complex. P450 eryF is thus a structurally defined model for the catalytic turnover of multiply bound substrates proposed to occur with CYP3A4. In view of its large active site and defined structure, catalytically active P450 eryF mutants are also attractive templates for the engineering of novel P450 activities. P450 eryF1 (CYP107A1) catalyzes the stereospecific 6(S)-hydroxylation of deoxyerythronolide B (6-DEB) in the biosynthesis of erythromycin by Saccharopolyspora erythraea ( Fig. 1) (1-3). The genetic manipulation of macrocyclic antibiotic biosynthetic pathways, including that of erythromycin, is currently under investigation as a route for the production of novel antibiotics (4). Hydroxylations catalyzed by P450 enzymes play key roles in these biosynthetic pathways, and modification of the substrate and regiospecificity of appropriate P450 enzymes is therefore of considerable interest. In an early example, targeted disruption of the gene encoding P450 eryF in S. erythraea yielded a strain that was unable to hydroxylate 6-DEB and which therefore produced 6-deoxyerythromycin (3).P450 eryF , a soluble 45-kDa protein, has been crystallized, and its structure has been determined in complexes with both the natural substrate 6-DEB and alternative ligands (5, 6). Two endogenous proteins able to provide electrons for turnover of P450 eryF have been cloned and expressed (7,8), although spinach ferredoxin and FNR function as acceptable surrogate electron donors (9). The structure of P450 eryF reveals two particularly interesting features of the enzyme. One is that the active site is much larger than the active sites of the other structurally defined bacterial P450 enzymes, as expected from the size of its macrocyclic substrate. The second is that the highly conserved threonine, Thr 252 in P450 cam (CYP101), is replaced in P450 eryF by Ala 245 (5, 6). The conserved threonine is thought to be required for dioxygen bond cleavage in the activation of molecular oxy...

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“…The possibility remains, however, that the altered residue had undergone 0-demethylation. From sequence homology alignments a highly conserved threonine corresponding to that in position 252 of P450cam has been identified in most known P450 sequences, an exception being P45OEryF, which has a valine instead (23)(24)(25)(26). The conserved threonine of several mammalian P450 isoforms has been mutated to examine its role in catalysis.…”

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confidence: 99%

Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4.

Vaz

Pernecky²,

Raner³

et al. 1996

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

213

165

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Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent ironoxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder [569][570][571][572][573][574][575][576][577][578][579][580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 jeleted [2B4 (A2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (A2-27), and 2B4 (A2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6-to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as-alternative oxygenating agents with the numerous isoforms of this versatile catalyst.The cytochrome P450 (P450) gene superfamily includes numerous hemoproteins involved in the metabolism of steroid hormones, vitamins, retinoids, eicosanoids, and other physiologically occurring compounds, as well as of an almost unlimited variety of drugs, pesticides, carcinogens, and other xenobiotics (1). These versatile catalysts are thought to use as an active oxidant a hypervalent iron-oxenoid species comparable to compound I of peroxidases (2-5). Although the identity of this oxidant in P450-catalyzed reactions has not been rigorously established, mechanistic evi...

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Substrate specificity of 6-deoxyerythronolide B hydroxylase, a bacterial cytochrome P450 of erythromycin A biosynthesis

Cited by 85 publications

References 33 publications

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

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

An A245T Mutation Conveys on Cytochrome P450eryF the Ability to Oxidize Alternative Substrates

Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4.

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