Self-Sufficient Class VII Cytochromes P450: From Full-Length Structure to Synthetic Biology Applications

Correddu, Danilo; Nardo, Giovanna Di; Gilardi, Gianfranco

doi:10.1016/j.tibtech.2021.01.011

Cited by 36 publications

(25 citation statements)

References 81 publications

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“…Enzymes belonging to class I to VI require a separate reductase that must interact using a specific mechanism of protein–protein interaction. On the other hand, classes from VII to X are actually self-sufficient [ 20 , 21 , 22 ], meaning that they are composed by a single polypeptide chain that includes a reductase and P450 domain [ 23 ]. As for the P450 of Class II, cytochrome P450 reductase (CPR) is the only physiological partner for many different P450s and how CPR is able to mediate electron transfer to a variety of different electron acceptors remains an open question [ 8 ].…”

Section: Introductionmentioning

confidence: 99%

Molecular Lego of Human Cytochrome P450: The Key Role of Heme Domain Flexibility for the Activity of the Chimeric Proteins

Catucci

Ciaramella

Nardo

et al. 2022

IJMS

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The cytochrome P450 superfamily are heme-thiolate enzymes able to carry out monooxygenase reactions. Several studies have demonstrated the feasibility of using a soluble bacterial reductase from Bacillus megaterium, BMR, as an artificial electron transfer partner fused to the human P450 domain in a single polypeptide chain in an approach known as ‘molecular Lego’. The 3A4-BMR chimera has been deeply characterized biochemically for its activity, coupling efficiency, and flexibility by many different biophysical techniques leading to the conclusion that an extension of five glycines in the loop that connects the two domains improves all the catalytic parameters due to improved flexibility of the system. In this work, we extend the characterization of 3A4-BMR chimeras using differential scanning calorimetry to evaluate stabilizing role of BMR. We apply the ‘molecular Lego’ approach also to CYP19A1 (aromatase) and the data show that the activity of the chimeras is very low (<0.003 min−1) for all the constructs tested with a different linker loop length: ARO-BMR, ARO-BMR-3GLY, and ARO-BMR-5GLY. Nevertheless, the fusion to BMR shows a remarkable effect on thermal stability studied by differential scanning calorimetry as indicated by the increase in Tonset by 10 °C and the presence of a cooperative unfolding process driven by the BMR protein domain. Previously characterized 3A4-BMR constructs show the same behavior of ARO-BMR constructs in terms of thermal stabilization but a higher activity as a function of the loop length. A comparison of the ARO-BMR system to 3A4-BMR indicates that the design of each P450-BMR chimera should be carefully evaluated not only in terms of electron transfer, but also for the biophysical constraints that cannot always be overcome by chimerization.

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Section: Introductionmentioning

confidence: 99%

Molecular Lego of Human Cytochrome P450: The Key Role of Heme Domain Flexibility for the Activity of the Chimeric Proteins

Catucci

Ciaramella

Nardo

et al. 2022

IJMS

Self Cite

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“…[201] Urlacher et al extended use of the P450-ADH cascade to synthesise (þ)-nootkatone (66), a high-value sesquiterpenoid. [202] The group selected P450 BM3 mutant BM3-A1 to catalyse the allylic hydroxylation of (þ)-valencene (67) to produce cis-nootkatol (68) and transnootkatol (69), which were subsequently oxidised to 66 by an unselective ADH mutant ADH-21, in yields reaching 360 mg L À 1 after 20 hours. [202] Similarly to work by Gröger et al, the authors used the oxidised cofactor NADP + , which was reduced in situ by ADH using 2-butanol as sacrificial substrate (Figure 9B).…”

Section: Biocatalytic Cascadesmentioning

confidence: 99%

Oxygenating Biocatalysts for Hydroxyl Functionalisation in Drug Discovery and Development

Charlton

Hayes

2022

ChemMedChem

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CÀ H oxyfunctionalisation remains a distinct challenge for synthetic organic chemists. Oxygenases and peroxygenases (grouped here as "oxygenating biocatalysts") catalyse the oxidation of a substrate with molecular oxygen or hydrogen peroxide as oxidant. The application of oxygenating biocatalysts in organic synthesis has dramatically increased over the last decade, producing complex compounds with potential uses in the pharmaceutical industry. This review will focus on hydroxyl functionalisation using oxygenating biocatalysts as a tool for drug discovery and development. Established oxygenating biocatalysts, such as cytochrome P450s and flavin-dependent monooxygenases, have widely been adopted for this purpose, but can suffer from low activity, instability or limited substrate scope. Therefore, emerging oxygenating biocatalysts which offer an alternative will also be covered, as well as considering the ways in which these hydroxylation biotransformations can be applied in drug discovery and development, such as latestage functionalisation (LSF) and in biocatalytic cascades.

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“…A short electron transport chain shuttles electrons from either NADH or NADPH to the oxygenase. In bacteria, these are usually two-or three-component systems in which an FAD-containing reductase reduces the P450 either directly or indirectly via a ferredoxin (Figure 2) [13], although single component systems also occur [14]. As covered in detail elsewhere [15], the heme iron in the resting state enzyme is in a low spin, ferric state.…”

Section: Structure and Mechanism Of P450smentioning

confidence: 99%

“…Intriguingly, modulating the ratio of components in a myxobacterial CYP260A1 system affected the product profiles of the P450 [47]. Related to this, engineering of P450-redox partner fusions, analogous to Class VII systems, may enable more efficient biocatalytic conversion [14]. For example, the P450BM3 fusion protein has been employed in a synthetic peroxygenase system to regio-selectively O-demethylate several aromatic ethers [48].…”

Section: Metabolic Engineeringmentioning

confidence: 99%

Cytochromes P450 in the biocatalytic valorization of lignin

Wolf

Hinchen

DuBois

et al. 2022

Current Opinion in Biotechnology

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Self-Sufficient Class VII Cytochromes P450: From Full-Length Structure to Synthetic Biology Applications

Cited by 36 publications

References 81 publications

Molecular Lego of Human Cytochrome P450: The Key Role of Heme Domain Flexibility for the Activity of the Chimeric Proteins

Molecular Lego of Human Cytochrome P450: The Key Role of Heme Domain Flexibility for the Activity of the Chimeric Proteins

Oxygenating Biocatalysts for Hydroxyl Functionalisation in Drug Discovery and Development

Cytochromes P450 in the biocatalytic valorization of lignin

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