Direct Conversion of Ethane to Ethanol by Engineered Cytochrome P450 BM3

Meinhold, Peter; Peters, Matthew W.; Chen, Michael M. Y.; Takahashi, Katsuyuki; Arnold, Frances H.

doi:10.1002/cbic.200500261

Cited by 153 publications

(125 citation statements)

References 28 publications

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“…Exciting recent results with the bacterial enzyme P450 BM3 have illustrated the potential of directed evolution for engineering P450s that use artificial oxygen donors (Glieder et al, 2002;Meinhold et al, 2005). Recently, P450 BM3 has been engineered by directed evolution to produce the authentic human metabolites of propranolol (Otey et al, 2006).…”

mentioning

confidence: 99%

Engineering of Cytochrome P450 3A4 for Enhanced Peroxide-Mediated Substrate Oxidation Using Directed Evolution and Site-Directed Mutagenesis

Kumar¹,

Liu²,

Halpert³

2006

Drug Metab Dispos

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ABSTRACT:CYP3A4 has been subjected to random and site-directed mutagenesis to enhance peroxide-supported metabolism of several substrates. Initially, a high-throughput screening method using whole cell suspensions was developed for H 2 O 2 -supported oxidation of 7-benzyloxyquinoline. Random mutagenesis by error-prone polymerase chain reaction and activity screening yielded several CYP3A4 mutants with enhanced activity. L216W and F228I showed a 3-fold decrease in K m, HOOH and a 2.5-fold increase in k cat /K m, HOOH compared with CYP3A4. Subsequently, T309V and T309A were created based on the observation that T309V in CYP2D6 has enhanced cumene hydroperoxide (CuOOH)-supported activity. T309V and T309A showed a >6-and 5-fold higher k cat /K m, CuOOH than CYP3A4, respectively. Interestingly, L216W and F228I also exhibited, respectively, a >4-and a >3-fold higher k cat /K m, CuOOH than CYP3A4. Therefore, several multiple mutants were constructed from rationally designed and randomly isolated mutants; among them, F228I/T309A showed an 11-fold higher k cat /K m, CuOOH than CYP3A4. Addition of cytochrome b 5 , which is known to stimulate peroxide-supported activity, enhanced the k cat /K m, CuOOH of CYP3A4 by 4-to 7-fold. When the mutants were tested with other substrates, T309V and T433S showed enhanced k cat /K m, CuOOH with 7-benzyloxy-4-(trifluoromethyl)coumarin and testosterone, respectively, compared with CYP3A4. In addition, in the presence of cytochrome b 5 , T433S has the potential to produce milligram quantities of 6␤-hydroxytestosterone through peroxide-supported oxidation. In conclusion, a combination of random and site-directed mutagenesis approaches yielded CYP3A4 enzymes with enhanced peroxide-supported metabolism of several substrates.

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confidence: 99%

Engineering of Cytochrome P450 3A4 for Enhanced Peroxide-Mediated Substrate Oxidation Using Directed Evolution and Site-Directed Mutagenesis

Kumar¹,

Liu²,

Halpert³

2006

Drug Metab Dispos

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“…Key to the activity of sMMO is the proposed diiron µ-oxo active site ( Figure 1) [81]. More recently Meinhold et al have shown engineered variants of P450 BM3 to be active for the fast oxidation of ethane to ethanol, with turnover frequencies mol ethanol¨m ol protein´1¨h´1 of 500 reported, at an NADPH oxidation rate of 31,200 h´1 [82]. The active site for P450 is shown in Figure 2.…”

Section: Enzymatic Approachesmentioning

confidence: 99%

“…Crucially, sMMO was able to avoid production of deeper oxidation products acetic acid and CO2. Key to the activity of sMMO is the proposed diiron μ-oxo active site (Figure 1 [82]. The active site for P450 is shown in Figure 2.…”

Section: Enzymatic Approachesmentioning

confidence: 99%

An Overview of Recent Advances of the Catalytic Selective Oxidation of Ethane to Oxygenates

2016

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Abstract:The selective partial oxidation of short chain alkanes is a key challenge within catalysis research. Direct ethane oxidation to oxygenates is a difficult aim, but potentially rewarding, and it could lead to a paradigm shift in the supply chain of several bulk chemicals. Unfortunately, low C-H bond reactivity and kinetically labile products are just some reasons affecting the development and commercialisation of such processes. Research into direct ethane oxidation is therefore disparate, with approaches ranging from oxidation in the gas phase at high temperatures to enzyme catalysed hydroxylation under ambient conditions. Furthermore, in overcoming the barrier posed by the chemically inert C-H bond a range of oxidants have been utilised. Despite years of research, this remains an intriguing topic from both academic and commercial perspectives. Herein we describe some recent developments within the field of catalytic ethane oxidation focusing on the formation of oxygenated products, whilst addressing the key challenges which are still to be overcome.

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“…Characteristics such as thermostability (10), solvent tolerance (25), enantioselectivity (7), substrate specificity (8), and catalytic efficiency (9) have all been altered to better adapt enzymes for specific purposes. Several examples applying molecular evolution to CYP enzymes have also been described in the literature, including the modification of the substrate specificity of the CYP BM3 from Bacillus megaterium (18,33). The wild-type CYP BM3 hydroxylates long-chain fatty acids predominantly in the -2 position.…”

mentioning

confidence: 99%

“…Site-directed mutagenesis provided mutants able to hydroxylate shorter-chain fatty acids and cyclic hydrocarbons. Multiple rounds of molecular evolution were necessary to adapt the enzyme to catalyze the direct hydroxylation of first octane, then propane, and finally ethane as a substrate (18).…”

mentioning

confidence: 99%

Biocatalytic Conversion of Avermectin to 4″-Oxo-Avermectin: Improvement of Cytochrome P450 Monooxygenase Specificity by Directed Evolution

Trefzer

Jungmann

Molnár

et al. 2007

Appl Environ Microbiol

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Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4؆-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4؆-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.Emamectin benzoate is a potent semisynthetic insecticide used to control many agriculturally important pests. However, wider application of emamectin has been hindered by the high cost of its chemical synthesis, which involves regioselective oxidation of the 4Љ alcohol of the natural product avermectin, followed by the reductive amination of the resulting key intermediate 4Љ-oxo-avermectin (see Fig. 1). Achievement of high regioselectivity in the oxidation reaction requires the costly protection and deprotection of the very reactive allylic hydroxyl function at C-5 of the avermectin molecule.As an alternative to chemical synthesis, microbial conversion has been used successfully in other regioselective oxidation reactions to bypass complex protection and deprotection strategies. Many microbial oxidative bioconversion reactions are carried out by dehydrogenases (4) or oxidases (3, 11) while some use cytochrome P450 monooxygenases (CYPs) (12a, 12b, 15-17). The cytochrome P450 family is a large class of enzymes that catalyze hydroxylation, epoxidation, and demethylation reactions. Class II CYPs from eukaryotes accept electrons from NADPH via a NADPH-cytochrome P450 reductase, whereas class I CYPs in prokaryotes use distinct ferredoxins and ferredoxin reductases for electron transfer from NADH. Biotransformations using CYPs have to regenerate the cofactor, which can be achieved cost-effectively on an industrial scale by using metabolically active whole cells as biocatalysts. Examples of CYP-catalyzed biotransformations include the 11␤-hydroxylation of Reichstein S to hydrocortisone (2) and the microbial hydroxylation of zofimarin, a sordarin-related antibiotic (32).Recently, we have reported the isolation and characterization of 17 CYP enzymes, collectively named the Ema CYPs, capable of regioselectively oxidizing avermectin to 4Љ-oxo-avermectin (13,19,20). The Ema CYPs were isolated from closely related Streptomyces spec...

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Direct Conversion of Ethane to Ethanol by Engineered Cytochrome P450 BM3

Cited by 153 publications

References 28 publications

Engineering of Cytochrome P450 3A4 for Enhanced Peroxide-Mediated Substrate Oxidation Using Directed Evolution and Site-Directed Mutagenesis

Engineering of Cytochrome P450 3A4 for Enhanced Peroxide-Mediated Substrate Oxidation Using Directed Evolution and Site-Directed Mutagenesis

An Overview of Recent Advances of the Catalytic Selective Oxidation of Ethane to Oxygenates

Biocatalytic Conversion of Avermectin to 4″-Oxo-Avermectin: Improvement of Cytochrome P450 Monooxygenase Specificity by Directed Evolution

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