Cytochrome P-450terp. Isolation and purification of the protein and cloning and sequencing of its operon.

Peterson, Julian A.; Lu, Jui-Yun; Geisselsoder, Janet; Graham-Lorence, Sandra E.; Carmona, Cynthia L.; Witney, Frank; Lorence, Matthew C.

doi:10.1016/s0021-9258(19)49697-x

Cited by 98 publications

(16 citation statements)

References 78 publications

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“…Protein Expression, Purification, and NMR Sample Preparation. Preliminary two-dimensional 1 H NMR experiments on Tdx o were performed using samples of Tdx purified from Pseudomonas grown using R-terpineol as sole carbon source (2). However, to obtain sufficient Tdx for extensive NMR and biophysical studies, overexpression in E. coli was necessary.…”

Section: Methodsmentioning

confidence: 99%

“…The camphor hydroxylase enzyme system consists of three proteins, putidaredoxin reductase (PdR), putidaredoxin (Pdx), 1 and CYP101. Recently, a similar hydroxylase has been identified in another strain of Pseudomonas capable of using R-terpineol as sole carbon source (2,3). Like the camphor hydroxylase, the terpineol hydroxylase system consists of three proteins: terpredoxin reductase (TdR), the Fe 2 S 2 ferredoxin † This work was supported in part by a grant from the National Institutes of Health (GM44191, T.C.P.).…”

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confidence: 99%

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A Model for the Solution Structure of Oxidized Terpredoxin, a Fe₂S₂ Ferredoxin from Pseudomonas^,

Pochapsky

1999

Biochemistry

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Terpredoxin (Tdx) is a 105-residue bacterial ferredoxin consisting of a single polypeptide chain and a single Fe2S2 prosthetic group. Tdx was first identified in a strain of Pseudomonas sp. capable of using alpha-terpineol as sole carbon source. The Tdx gene, previously cloned from the plasmid-encoded terp operon, that carries genes encoding for proteins involved in terpineol catabolism, has been subcloned and expressed as the holoprotein in E. coli. Physical characterization of the expressed Tdx has been performed, and a model for the solution structure of oxidized Tdx (Tdxo) has been determined. High-resolution homo- and heteronuclear NMR data have been used for structure determination in diamagnetic regions of the protein. The structure of the metal binding site (which cannot be determined directly by NMR methods due to paramagnetic broadening of resonances) was modeled using restraints obtained from a crystal structure of the homologous ferredoxin adrenodoxin (Adx) and loose restraints determined from paramagnetic broadening patterns in NMR spectra. Essentially complete 1H and 15N NMR resonance assignments have been made for the diamagnetic region of Tdxo (ca. 80% of the protein). A large five-stranded beta-sheet and a smaller two-stranded beta-sheet were identified, along with three alpha-helices. A high degree of structural homology was observed between Tdx and two other ferredoxins with sequence and functional homology to Tdx for which structures have been determined, Adx and putidaredoxin (Pdx), a homologous Pseudomonas protein. 1H/2H exchange rates for Tdx backbone NH groups were measured for both oxidation states and are rationalized in the context of the Tdx structure. In particular, an argument is made for the importance of the residue following the third ligand of the metal cluster (Arg49 in Tdx, His49 in Pdx, His56 in Adx) in modulating protein dynamics as a function of oxidation state. Some differences between Tdx and Pdx are detected by UV-visible spectroscopy, and structural differences at the C-terminal region were also observed. Tdx exhibits only 2% of the activity of Pdx in turnover assays performed using the reconstituted camphor hydroxylase system of which Pdx is the natural component.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

A Model for the Solution Structure of Oxidized Terpredoxin, a Fe₂S₂ Ferredoxin from Pseudomonas^,

Pochapsky

1999

Biochemistry

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“…P450terp is a bacterial enzyme (CYP108, Pseudomonas sp.) which catalyzes the oxidation of the monoterpene hydrocarbon a-terpineol (Co 1 H l 8 0) in the catabolic assimilation of this compound as a sole source of carbon and energy [24]. Like P450cam, P450terp is grouped with the class I P450s based on sequence homology and its requirement for an iron-sulfur protein as an electron donor.…”

Section: Introductionmentioning

confidence: 99%

Structure and function of cytochromes P450:a comparative analysis of three crystal structures

et al. 1995

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Based on this comparison we believe that all P450s will be found to possess the same tertiary structure. The ability to precisely predict other P450 substrate-contact residues is limited by the extreme structural heterogeneity in the substrate-recognition regions. The central I-helix structures of P450terp and P450BM-3 suggest a role for helix-associated solvent molecules as a source of catalytic protons, distinct from the mechanism for P450cam. We suggest that the P450 molecular dipole might aid in both redox-partner docking and proton recruitment for catalysis.

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“…P450tol shares 46% sequence identity with the P450terp from Pseudomonas aeruginosa. 11 E. coli (P450tol-1) co-expressing P450tol and its ferredoxin (Fdx) and ferredoxin reductase (FdR) showed an activity of 0.52 U g À1 cell dry weight (CDW) for the epoxidation of 3-bromo-styrene 1. To improve the activity, another three pairs of Fdx and FdR from different bacteria were co-expressed with P450tol (ESI, † Table S7).…”

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Asymmetric epoxidation of alkenes and benzylic hydroxylation with P450tol monooxygenase from Rhodococcus coprophilus TC-2

Adams

et al. 2014

Chem. Commun.

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P450tol monooxygenase was discovered as a unique and highly enantioselective enzyme for asymmetric epoxidation of some terminal alkenes containing electron-withdrawing groups and benzylic hydroxylation of several ethylbenzenes giving the corresponding useful and valuable products, such as (R)-2- and 3-substituted styrene oxides, (S)-4-substituted styrene oxides, and (S)-benzylic alcohols, in high ee.

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Cytochrome P-450terp. Isolation and purification of the protein and cloning and sequencing of its operon.

Cited by 98 publications

References 78 publications

A Model for the Solution Structure of Oxidized Terpredoxin, a Fe₂S₂ Ferredoxin from Pseudomonas^,

A Model for the Solution Structure of Oxidized Terpredoxin, a Fe₂S₂ Ferredoxin from Pseudomonas^,

Structure and function of cytochromes P450:a comparative analysis of three crystal structures

Asymmetric epoxidation of alkenes and benzylic hydroxylation with P450tol monooxygenase from Rhodococcus coprophilus TC-2

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Cytochrome P-450terp. Isolation and purification of the protein and cloning and sequencing of its operon.

Cited by 98 publications

References 78 publications

A Model for the Solution Structure of Oxidized Terpredoxin, a Fe2S2 Ferredoxin from Pseudomonas,

A Model for the Solution Structure of Oxidized Terpredoxin, a Fe2S2 Ferredoxin from Pseudomonas,

Structure and function of cytochromes P450:a comparative analysis of three crystal structures

Asymmetric epoxidation of alkenes and benzylic hydroxylation with P450tol monooxygenase from Rhodococcus coprophilus TC-2

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A Model for the Solution Structure of Oxidized Terpredoxin, a Fe₂S₂ Ferredoxin from Pseudomonas^,

A Model for the Solution Structure of Oxidized Terpredoxin, a Fe₂S₂ Ferredoxin from Pseudomonas^,