Peroxidase activity stabilization of cytochrome P450BM3 by rational analysis of intramolecular electron transfer

Vidal-Limón, Abraham; Águila, Sergio; Ayala, Marcela; Batista, Cesar Vicente Ferreira; Vázquez-Duhalt, Rafael

doi:10.1016/j.jinorgbio.2013.01.009

Cited by 46 publications

(39 citation statements)

References 68 publications

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“…Several mutagenesis approaches showed the possibility to transform conventional P450s into peroxygenases [59,61]. Therefore, peroxygenases have high potential; however, since elevated H 2 O 2 -concentrations are harmful for both, the enzyme and its heme-cofactor [58][59][60], stable variants [59,61] and variants rescuing the heme-cofactor are highly desired. Furthermore, alternative in situ H 2 O 2 -production methods [69,133] or feeding of low amounts of H 2 O 2 may be exploited.…”

Section: Discussionmentioning

confidence: 99%

“…However, P450 peroxygenases are also prone to inactivation due to the required high concentrations of hydrogen peroxide [58][59][60]. Mutagenesis approaches including directed evolution [61] and site-directed mutagenesis [59] showed successful stabilization and increased activity of peroxygenases.…”

Section: Mechanistic Considerationsmentioning

confidence: 99%

“…However, to a lower extent from 10 to 50 min −1 [61]. By a QM/MM-guided approach residues W96 and F405 in the 10-fold variant were identified to be oxidizable by H 2 O 2 , which allowed the design of a more stable variant retaining the activity (single or double variants: W96A, F405L or W96A/F405L) [59].…”

Section: Towards New Electron Sources For Cyp102a1mentioning

confidence: 99%

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Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

2017

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Section: Discussionmentioning

confidence: 99%

Section: Mechanistic Considerationsmentioning

confidence: 99%

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Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

2017

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“…P450BM3's suicide inactivation [197], a common process in heme peroxidases. From the QM-MM calculations they identified key residues in the second heme coordination sphere, aiming at reducing electron delocalization and obtaining a more stable enzyme against H 2 O 2 .…”

Section: Catalytic Rate Constant Enhancementmentioning

confidence: 99%

Molecular Modeling in Enzyme Design, Toward In Silico Guided Directed Evolution

Monza

Acebes

Lucas

et al. 2017

Directed Enzyme Evolution: Advances and Applications

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Directed evolution creates diversity in subsequent rounds of mutagenesis in the quest of increased protein stability, substrate binding and catalysis. Although this technique does not require any structural/mechanistic knowledge of the system, the frequency of improved mutations is usually low. For this reason, computational tools are increasingly used to focus the search in sequence space, enhancing the efficiency of laboratory evolution. In particular, molecular modeling methods provide a unique tool to grasp the sequence/structure/function relationship The final publication is available at Springer via https://link.springer.com/chapter/10.1007/978-3-319-50413-1_10/fulltext.html of the protein to evolve, with the only condition that a structural model is provided. With this book chapter, we tried to guide the reader through the state of art of molecular modeling, discussing their strengths, limitations and directions. In addition, we suggest a possible future template for in silico directed evolution where we underline two main points: a hierarchical computational protocol combining several different techniques, and a synergic effort between simulations and experimental validation. IntroductionBiotechnology needs catalysts that can work under harsh conditions, catalyze a broad range of substrates, generate maximum amount of product, and tolerate changes in the environment. Enzymes, which are biodegradable and reusable catalysts [1], in addition to remarkable reaction rates, can work in environmentally friendly pH and temperature ranges, and display control over stereochemistry and regioselectivity which makes them ideal for many applications [2,3]. When thinking about enzymes, people normally associate them to expressions such as "perfect catalysts" or "outstanding reaction rate". In fact, there are examples of enzymes that catalyze reactions at extremely high rates such as triose phosphate isomerase, superoxide dismutase or carbonic anhydrase [4]. These are often limited only by the rate of ligand diffusion into the active site (diffusion-controlled rate). Nevertheless, an extensive analysis by Bar-Even et al., of nearly 2000 enzymes, showed that the median maximal turnover rate value over all measured enzymes is about 10 s −1 nowhere close to the values of 10 5 or 10 6 normally associated with catalysts [5,6].So, it would appear that natural enzymes are "just good enough" for the function they must perform in a given organism [7]. One might conclude that if they had evolved to their optimum performance then trying to improve them (from a kinetic point of view) would be attempting the impossible. On the contrary, as seen by the distribution of reaction rates, k cat , most enzymes function at a lower rate than the diffusion-limit and thus, there is space to further increase their kinetic properties to meet industrial needs. Additionally, we need enzymes capable of catalyzing reactions for which no known enzymes exist, to work with different substrates and for particular conditions that are industrially...

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“…A variety of P450 mutants have been prepared by site-directed mutagenesis and directed evolution (random mutagenesis) for the development of artificial H 2 O 2 -dependent P450s [71][72][73][74][75]. Among them, mutants of P450BM3 showed relatively high peroxygenase activity.…”

Section: Engineering Of P450s For Construction Of Artificial H 2 O 2 mentioning

confidence: 99%

Monooxygenation of Small Hydrocarbons Catalyzed by Bacterial Cytochrome P450s

Okada

Watanabe

2015

Advances in Experimental Medicine and Biology

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Cytochrome P450s (P450s) catalyze the NAD(P)H/O2-dependent monooxygenation of less reactive organic molecules under mild conditions. The catalytic activity of bacterial P450s is very high compared with P450s isolated from animals and plants, and the substrate specificity of bacterial P450s is also very high. Accordingly, their catalytic activities toward nonnative substrates are generally low especially toward small hydrocarbons. However, mutagenesis approaches have been very successful for engineering bacterial P450s for the hydroxylation of small hydrocarbons. On the other hand, "decoy" molecules, whose structures are very similar to natural substrates, can be used to trick the substrate recognition of bacterial P450s, allowing the P450s to catalyze oxidation reactions of nonnative substrates without any substitution of amino acid residues in the presence of decoy molecules. Thus, the hydroxylation of small hydrocarbons such as ethane, propane, butane and benzene can be catalyzed by P450BM3, a long-alkyl-chain hydroxylase, using substrate misrecognition of P450s induced by decoy molecules. Furthermore, a number of H2O2-dependent bacterial P450s can catalyze the peroxygenation of a variety of nonnative substrates through a simple substrate-misrecognition trick, in which catalytic activities and enantioselectivity are dependent on the structure of decoy molecules.

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Peroxidase activity stabilization of cytochrome P450BM3 by rational analysis of intramolecular electron transfer

Cited by 46 publications

References 68 publications

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

Molecular Modeling in Enzyme Design, Toward In Silico Guided Directed Evolution

Monooxygenation of Small Hydrocarbons Catalyzed by Bacterial Cytochrome P450s

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