Modulation of the interaction between human P450 3A4 and B. megaterium reductase via engineered loops

Castrignanò, Silvia; D'Avino, Serena; Nardo, Giovanna Di; Catucci, Gianluca; Sadeghi, Sheila J.; Gilardi, Gianfranco

doi:10.1016/j.bbapap.2017.07.009

Cited by 16 publications

(12 citation statements)

References 46 publications

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“…In terms of loop length when the three different chimeras are compared, the 3A4-BMR-5GLY one showed a higher stability of the P450 module, a higher coupling efficiency, and also a higher cytochrome c reduction rate [ 34 ]. The loop length also positively impacted the binding affinity and cooperativity for testosterone ultimately leading to increased k cat [ 35 ]. Furthermore, hydrogen/deuterium exchange kinetics measured by FTIR revealed that 3A4-BMR-5GLY displayed higher exposure of the protons indicating that the surface area between the P450 and the BMR domain is more accessible to the solvent [ 36 ].…”

Section: Introductionmentioning

confidence: 99%

“…Finally, electrochemistry experiments performed in different cross-linking preparations showed that a more flexible immobilizing gel results in higher electrocatalysis, especially for the 3A4-BMR-5GLY chimera [ 36 ]. All the above mentioned data indicate that a connecting loop increased by five glycine residues is able to improve the stability of the protein and the coupling efficiency through a higher flexibility of the system [ 34 , 35 , 36 ]. Moreover, molecular modelling studies indicated that the chimeras between the CYP3A4 and BMR, the length of the loop is crucial for the overall flexibility of the system because a shorter loop is predicted to form a secondary structure that rigidifies the movement of the FMN domain in the process of docking to the heme proximal site of the P450 domain [ 35 ].…”

Section: Introductionmentioning

confidence: 99%

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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%

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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“…Further studies investigated the role of the heme domain-reductase domain connecting loop on electron transfer efficiency by addition of glycine residues. The data showed that the addition of five glycines increased the Vmax by two-fold for testosterone [77,78]. BM3's reductase domain was also fused to the bacterial P450 TxtE, which was shown to The Arnold group designed some of the first reported BM3 chimeras, where the BM3 heme domain was combined with the homologues, CYP102A2 and CYP102A3, as part of a promiscuous library which aimed to expand the catalytic repertoire for the metabolism of structurally diverse compounds [43].…”

Section: Production Of Human P450 Metabolitesmentioning

confidence: 99%

“…Further studies investigated the role of the heme domain-reductase domain connecting loop on electron transfer efficiency by addition of glycine residues. The data showed that the addition of five glycines increased the V max by two-fold for testosterone [ 77 , 78 ]. BM3’s reductase domain was also fused to the bacterial P450 TxtE, which was shown to be crucial in the thaxtomin pathway; a fusion with 14 amino acid linkers was found to reach a 100% turnover by 8 h with a coupling efficiency 2.2 times higher than standalone TxtE [ 79 ].…”

Section: Engineering Bm3 For Drug Metabolite Productionmentioning

confidence: 99%

A Promiscuous Bacterial P450: The Unparalleled Diversity of BM3 in Pharmaceutical Metabolism

Thistlethwaite

Jeffreys

Girvan

et al. 2021

IJMS

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CYP102A1 (BM3) is a catalytically self-sufficient flavocytochrome fusion protein isolated from Bacillus megaterium, which displays similar metabolic capabilities to many drug-metabolizing human P450 isoforms. BM3′s high catalytic efficiency, ease of production and malleable active site makes the enzyme a desirable tool in the production of small molecule metabolites, especially for compounds that exhibit drug-like chemical properties. The engineering of select key residues within the BM3 active site vastly expands the catalytic repertoire, generating variants which can perform a range of modifications. This provides an attractive alternative route to the production of valuable compounds that are often laborious to synthesize via traditional organic means. Extensive studies have been conducted with the aim of engineering BM3 to expand metabolite production towards a comprehensive range of drug-like compounds, with many key examples found both in the literature and in the wider industrial bioproduction setting of desirable oxy-metabolite production by both wild-type BM3 and related variants. This review covers the past and current research on the engineering of BM3 to produce drug metabolites and highlights its crucial role in the future of biosynthetic pharmaceutical production.

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“…Moreover, cytochromes P450 were found to be a valuable catalysts for lignin bioconversion [40]. Cytochromes P450 are known to be extremely powerful catalysts with broad substrate specificity [41,42] that can be exploited for pharmaceutical, environmental, and bionsensing applications [43], but except for the peroxygenase subgroup, their activity relies in the proper shuttling, coupling, and optimization of electrons from a reductase partner [44,45]. This review focuses on the structure/function relationship of a new family of peroxidases, named dye-decolorizing peroxidases (DyPs) that have demonstrated a role in lignin breakdown [46], through the use hydrogen peroxide as cosubstrate and high potential for biotechnological applications thanks to their presence in bacterial genomes [47,48] that facilitates heterologous production in Escherichia coli.…”

Section: Figmentioning

confidence: 99%

Biochemical features of dye‐decolorizing peroxidases: Current impact on lignin degradation

Catucci

Valetti

Sadeghi

et al. 2020

Biotech and App Biochem

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Dye-decolorizing peroxidases (DyP) were originally discovered in fungi for their ability to decolorize several different industrial dyes. DyPs catalyze the oxidation of a variety of substrates such as phenolic and nonphenolic aromatic compounds. Catalysis occurs in the active site or on the surface of the enzyme depending on the size of the substrate and on the existence of radical transfer pathways available in the enzyme. DyPs show the typical features of heme-containing enzymes with a Soret peak at 404-408 nm. They bind hydrogen peroxide that leads to the formation of the so-called Compound I, the key intermediate for catalysis. This then decays into Compound II yielding back Fe(III) at its resting state. Each catalytic cycle uses two electrons from suitable electron donors and generates two product molecules. DyPs are classified as a separate class of

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Modulation of the interaction between human P450 3A4 and B. megaterium reductase via engineered loops

Cited by 16 publications

References 46 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

A Promiscuous Bacterial P450: The Unparalleled Diversity of BM3 in Pharmaceutical Metabolism

Biochemical features of dye‐decolorizing peroxidases: Current impact on lignin degradation

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