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Tricistronic overexpression of cytochrome P450cam, putidaredoxin, and putidaredoxin reductase provides a useful cell-based catalytic system.

ABSTRACT: The catalytic turnover of cytochrome P450( cam ) from Pseudomonas putida requires two auxiliary reduction partners, putidaredoxin (Pd) and putidaredoxin reductase (PdR). We report the functional expression in Escherichia coli of tricistronic constructs consisting of P450( cam ) encoded by the first cistron and the auxiliary proteins, Pd and PdR by the second and the third. Transformed bacterial whole cells efficiently oxidized (1R)-(+)-camphor to 5-exo-hydroxycamphor and, interestingly, limonene to (-)-perillyl alcohol. These bioengineered E. coli cells possess a heterologous self-sufficient P450 catalytic system that may have advantages in terms of low cost and high yield for the production of fine chemicals.

SUBMITTER: Kim D

PROVIDER: S-EPMC2844094 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Tricistronic overexpression of cytochrome P450cam, putidaredoxin, and putidaredoxin reductase provides a useful cell-based catalytic system.

Kim Donghak D Ortiz de Montellano Paul R PR

Biotechnology letters 20090521 9

The catalytic turnover of cytochrome P450( cam ) from Pseudomonas putida requires two auxiliary reduction partners, putidaredoxin (Pd) and putidaredoxin reductase (PdR). We report the functional expression in Escherichia coli of tricistronic constructs consisting of P450( cam ) encoded by the first cistron and the auxiliary proteins, Pd and PdR by the second and the third. Transformed bacterial whole cells efficiently oxidized (1R)-(+)-camphor to 5-exo-hydroxycamphor and, interestingly, limonene ...[more]

PMID: 19458919

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Partial fusion of a cytochrome P450 system by carboxy-terminal attachment of putidaredoxin reductase to P450cam (CYP101A1).

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Project description:The importance of conformational dynamics to protein function is now well-appreciated. An outstanding question is whether they are involved in the effector role played by putidaredoxin (Pdx) in its reduction of the O2 complex of cytochrome P450cam (P450cam), an archetypical member of the cytochrome P450 superfamily. Recent studies have reported that binding of Pdx induces a conformational change from a closed to an open state of ferric P450cam, but a similar conformational change does not appear to occur for the ferrous, CO-ligated enzyme. To better understand the effector role of Pdx when binding the ferrous, CO-ligated P450cam, we applied 2D IR spectroscopy to compare the conformations and dynamics of the wild-type (wt) enzyme in the absence and presence of Pdx, as well as of L358P P450cam (L358P), which has served as a putative model for the Pdx complex. The CO vibrations of the Pdx complex and L358P report population of two conformational states in which the CO experiences distinct environments. The dynamics among the CO frequencies indicate that the energy landscape of substates within one conformation are reflective of the closed state of P450cam, and for the other conformation, differ from the free wt enzyme, but are equivalent between the Pdx complex and L358P. The two states co-populated by the Pdx complex are postulated to reflect a loosely bound encounter complex and a more tightly bound state, as is commonly observed for the dynamic complexes of redox partners. Significantly, this study shows that the binding of Pdx to ferrous, CO-ligated P450cam does perturb the conformational ensemble in a way that might underlie the effector role of Pdx.

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Detection of a high-barrier conformational change in the active site of cytochrome P450cam upon binding of putidaredoxin.

Project description:The orientation of the substrate camphor in the active site of reduced CO-bound cytochrome P450cam (CYP101) as a function of reduced putidaredoxin (Pdxr) addition has been examined by NMR using perdeuterated CYP101 and perdeuterated Pdx as well as isotopically labeled d-camphor. This permits the 1H resonances of CYP101-bound camphor to be observed without interference from the signals of CYP101 or Pdx and confirms assignments of the methyl signals of camphor in the bound form. The Cys4Fe2S2 ferredoxin Pdx is the physiological redox partner and effector of CYP101. The addition of Pdx to the reduced CYP101-camphor-CO complex results in a conformational selection that is slow on the chemical shift time scale with spectral effects observed primarily at the 8-CH3 group of the camphor. The camphor signals are ring current shifted by the heme, and for the 9- and 10-CH3 resonances, these shifts are reasonably well predicted by ring current calculations from the crystal structure of CO-bound CYP101. However, in the absence of Pdx, the 8-CH3 resonance of CYP101-bound camphor is observed at considerably higher field than predicted. Dynamic simulations using ring current shift restraints generated a structure with low chemical shift violations in which the hydrogen bond between the camphor carbonyl oxygen and the OH of Tyr96 is lost, and an expansion of the active site takes place that permits reorientation of the camphor within the active site.

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Solution NMR structure of putidaredoxin-cytochrome P450cam complex via a combined residual dipolar coupling-spin labeling approach suggests a role for Trp106 of putidaredoxin in complex formation.

Project description:The 58-kDa complex formed between the [2Fe-2S] ferredoxin, putidaredoxin (Pdx), and cytochrome P450cam (CYP101) from the bacterium Pseudomonas putida has been investigated by high-resolution solution NMR spectroscopy. Pdx serves as both the physiological reductant and effector for CYP101 in the enzymatic reaction involving conversion of substrate camphor to 5-exo-hydroxycamphor. In order to obtain an experimental structure for the oxidized Pdx-CYP101 complex, a combined approach using orientational data on the two proteins derived from residual dipolar couplings and distance restraints from site-specific spin labeling of Pdx has been applied. Spectral changes for residues in and near the paramagnetic metal cluster region of Pdx in complex with CYP101 have also been mapped for the first time using (15)N and (13)C NMR spectroscopy, leading to direct identification of the residues strongly affected by CYP101 binding. The new NMR structure of the Pdx-CYP101 complex agrees well with results from previous mutagenesis and biophysical studies involving residues at the binding interface such as formation of a salt bridge between Asp38 of Pdx and Arg112 of CYP101, while at the same time identifying key features different from those of earlier modeling studies. Analysis of the binding interface of the complex reveals that the side chain of Trp106, the C-terminal residue of Pdx and critical for binding to CYP101, is located across from the heme-binding loop of CYP101 and forms non-polar contacts with several residues in the vicinity of the heme group on CYP101, pointing to a potentially important role in complex formation.

| S-EPMC5319408 | biostudies-literature

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