Engineered Alkane‐Hydroxylating Cytochrome P450<sub>BM3</sub> Exhibiting Nativelike Catalytic Properties

Fasan, Rudi; Chen, Mike M.; Crook, Nathan; Arnold, Frances H.

doi:10.1002/ange.200702616

Cited by 120 publications

(127 citation statements)

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“…The strains and plasmids used in this study are listed in Table 1. Luria-Bertani broth (23) and modified M9 medium with 1.5% yeast extract (8) supplemented with appropriate antibiotics, or E2 (15) and M9 (17) minimal media supplemented with carbon sources, were used for growth. All cultures were grown aerobically at 30°C (P. putida) or 37°C (Escherichia coli).…”

Section: Methodsmentioning

confidence: 99%

“…Recent studies have shown that high activities on small alkanes can be obtained by engineering bacterial P450 enzymes such as P450cam (CYP101; camphor hydroxylase) and P450 BM3 (CYP102A; a fatty acid hydroxylase) (8,36). The resulting enzymes, however, hydroxylate propane and higher alkanes primarily at the more energetically favorable subterminal positions; highly selective terminal hydroxylation is difficult to achieve by engineering a subterminal hydroxylase (22).…”

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confidence: 99%

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In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

Koch

Chen

Beilen

et al. 2009

Appl Environ Microbiol

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Enzymes of the AlkB and CYP153 families catalyze the first step in the catabolism of medium-chain-length alkanes, selective oxidation of the alkane to the 1-alkanol, and enable their host organisms to utilize alkanes as carbon sources. Small, gaseous alkanes, however, are converted to alkanols by evolutionarily unrelated methane monooxygenases. Propane and butane can be oxidized by CYP enzymes engineered in the laboratory, but these produce predominantly the 2-alkanols. Here we report the in vivo-directed evolution of two mediumchain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from Pseudomonas putida GPo1 and the class II-type soluble CYP153A6 from Mycobacterium sp. strain HXN-1500, to enhance their activity on small alkanes. We established a P. putida evolution system that enables selection for terminal alkane hydroxylase activity and used it to select propane-and butane-oxidizing enzymes based on enhanced growth complementation of an adapted P. putida GPo12(pGEc47⌬B) strain. The resulting enzymes exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation. This in vivo evolution system could be useful for directed evolution of enzymes that function efficiently to hydroxylate small alkanes in engineered hosts.Microbial utilization and degradation of alkanes was discovered almost a century ago (27). Since then, several enzyme families capable of hydroxylating alkanes to alkanols, the first step in alkane degradation, have been identified and categorized based on their preferred substrates (30). The soluble and particulate methane monooxygenases (sMMO and pMMO) and the related propane monooxygenase and butane monooxygenase (BMO) are specialized on gaseous small-chain alkanes (C 1 to C 4 ), while medium-chain (C 5 to C 16 ) alkane hydroxylation seems to be the domain of the CYP153 and AlkB enzyme families.Conversion of C 1 to C 4 alkanes to alkanols is of particular interest for producing liquid fuels or chemical precursors from natural gas. The MMO-like enzymes that catalyze this reaction in nature, however, exhibit limited stability or poor heterologous expression (30) and have not been suitable for use in a recombinant host that can be engineered to optimize substrate or cofactor delivery. Alkane monooxygenases often cometabolize a wider range of alkanes than those which support growth (12). We wished to determine whether it is possible to engineer a medium-chain alkane monooxygenase to hydroxylate small alkanes, thereby circumventing difficulties associated with en-

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

Koch

Chen

Beilen

et al. 2009

Appl Environ Microbiol

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“…Activity toward propane, ethane, and methane have not been reported for any naturally-occurring cytochrome P450, yet we have shown that highly proficient small-alkane P450 hydroxylases are possible and, indeed, easily accessible by mutation from existing P450s. One laboratory-evolved enzyme, P450 PMO (PMO) 1 derived from the fatty acid hydroxylase CYP102A1 from Bacillus megaterium (BM3), inserts oxygen primarily at the 2-position of propane to generate 2-propanol. We also found that vengineered enzymes derived from the newly-characterized longer-chain alkane hydroxylase CYP153 family can also hydroxylate propane and butane and do so primarily at the energetically disfavored terminal carbon (1-position).…”

Section: Detailed Report Aim 1 Determine Kinetic and Stabilities Ofmentioning

confidence: 99%

“…These variants, such as PMO, all exhibit a spin-shift in the presence of propane. 1 In order to achieve methane hydroxylation under turnover conditions we will need to evolve A6 to bind methane sufficiently to activate the catalytic cycle.…”

Section: A6 Substrate Binding Characterizationmentioning

confidence: 99%

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Structural and Kinetic Studies of Novel Cytochrome P450 Small-Alkane Hydroxylases

Arnold¹

2012

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Executive SummaryThe goals of this project are to investigate (1) the kinetics and stabilities of engineered cytochrome P450 (P450) small alkane hydroxylases and their evolutionary intermediates, (2) the structural basis for catalytic proficiency on small alkanes of these engineered P450s, and (3) the changes in redox control resulting from protein engineering. To reach these goals, we have established new methods for determining the kinetics and stabilities of multicomponent P450s such as CYP153A6. Using these, we were able to determine that CYP153A6 is proficient for hydroxylation of alkanes as small as ethane, an activity that has never been observed previously in any natural P450. To elucidate the structures of the engineered P450s, we obtained x-ray diffraction data for two variants in the P450 PMO (propane monooxygenase) lineage and a preliminary structure for the most evolved variant. This structure shows changes in the substrate binding regions of the enzyme and a reduction in active site volume that are consistent with the observed changes in substrate specificity from fatty acids in the native enzyme to small alkanes in P450 PMO . We also constructed semi-rational designed libraries mutating only residues in the enzyme active site that in one round of mutagenesis and screening produced variants that achieved nearly half of the activity of the most evolved enzymes of the P450 PMO lineage. Finally, we found that changes in redox properties of the laboratory-evolved P450 alkane hydroxylases did not reflect the improvement in their electron transfer efficiency. The heme redox potential remained constant throughout evolution, while activity increased and coupling efficiency improved from 10% to 90%. The lack of correlation between heme redox potential and enzyme activity and coupling efficiency led us to search for other enzyme properties that could be better predictors for activity towards small alkanes, specifically methane. We investigated the oxidation potential of the radical oxidants of various P450s directly using a chemical approach to generate the radical in situ. This resulted in the first report of direct methane to methanol conversion by a heme porphyrin catalyst using the soluble P450 from Mycobacterium sp, CYP153A6.

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Biocatalytic Asymmetric Oxidations in Stereoselective Synthesis

Schallmey

Marı́a

Bracco

2013

Stereoselective Synthesis of Drugs and Natural Products

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Enzyme‐catalyzed oxidations represent a versatile approach for the production of optically active building blocks and natural products. Moreover, enzymatic reaction conditions are often mild and with high atom economy, thus, enabling the basis for future environmentally friendly chemistry. As a result, several biocatalytic oxidative processes have already been implemented on an industrial scale as well, clearly showing that modern biocatalysis can compete economically with other chemical‐based processes. This chapter thoroughly describes the different types of oxidative enzymes that can be found in nature, namely, dehydrogenases, oxygenases, oxidases, and peroxidases, with emphasis on their mechanism and occurrence. Furthermore, several outstanding examples of biocatalytic applications of these enzymes are given—either by means of recombinant whole cells or isolated enzymes—involving the production of different drugs and natural products in which a catalytic step is catalyzed by these oxidative enzymes.

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Engineered Alkane‐Hydroxylating Cytochrome P450_BM3 Exhibiting Nativelike Catalytic Properties

Cited by 120 publications

References 46 publications

In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

Structural and Kinetic Studies of Novel Cytochrome P450 Small-Alkane Hydroxylases

Biocatalytic Asymmetric Oxidations in Stereoselective Synthesis

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Engineered Alkane‐Hydroxylating Cytochrome P450BM3 Exhibiting Nativelike Catalytic Properties

Cited by 120 publications

References 46 publications

In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

Structural and Kinetic Studies of Novel Cytochrome P450 Small-Alkane Hydroxylases

Biocatalytic Asymmetric Oxidations in Stereoselective Synthesis

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Engineered Alkane‐Hydroxylating Cytochrome P450_BM3 Exhibiting Nativelike Catalytic Properties