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/anie.200702616

Cited by 225 publications

(134 citation statements)

References 46 publications

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“…The highest biocatalyst utilization of P450s has been reported for CYP102A1-catalyzed hydroxylation of myristic acid [27] and propane oxidation. [28] With TNNs of 30 000 and 45 000, respectively, they are one magnitude higher than the TTNs achieved here for CYP106A2. As the applicability of an enzymatic process is determined by its overall costs, a rather low biocatalyst utilization is not automatically prohibitive for a large-scale application.…”

Section: Discussioncontrasting

confidence: 53%

Towards Preparative Scale Steroid Hydroxylation with Cytochrome P450 Monooxygenase CYP106A2

et al. 2010

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Cytochrome P450 monooxygenases are of outstanding interest for the synthesis of pharmaceuticals and fine chemicals, due to their ability to hydroxylate C--H bonds mainly in a stereo- and regioselective manner. CYP106A2 from Bacillus megaterium ATCC 13368, one of only a few known bacterial steroid hydroxylases, enables the oxidation of 3-keto-4-ene steroids mainly at position 15. We expressed this enzyme together with the electron-transfer partners bovine adrenodoxin and adrenodoxin reductase in Escherichia coli. Additionally an enzyme-coupled cofactor regeneration system was implemented by expressing alcohol dehydrogenase from Lactobacillus brevis. By studying the conversion of progesterone and testosterone, the bottlenecks of these P450-catalyzed hydroxylations were identified. Substrate transport into the cell and substrate solubility turned out to be crucial for the overall performance. Based on these investigations we developed a new concept for CYP106A2-catalyzed steroid hydroxylations by which the productivity of progesterone and testosterone conversion could be increased up to 18-fold to yield an absolute productivity up to 5.5 g L(-1) d(-1). Product extraction with absorber resins allowed the recovery of quantitative amounts of 15beta-OH-progesterone and 15beta-OH-testosterone and also the reuse of the biocatalyst.

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Section: Discussioncontrasting

confidence: 53%

Towards Preparative Scale Steroid Hydroxylation with Cytochrome P450 Monooxygenase CYP106A2

et al. 2010

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“…[44] In conclusion, the two crystal structures reported in this paper give contrasting perspectives on how the substitution of proline into an enzyme can alter catalytic properties. In A330P, dramatic but localised changes impact the active site.…”

mentioning

confidence: 96%

Structural Basis for the Properties of Two Single‐Site Proline Mutants of CYP102A1 (P450_BM3)

et al. 2010

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The crystal structures of the haem domains of Ala330Pro and Ile401Pro, two single-site proline variants of CYP102A1 (P450(BM3)) from Bacillus megaterium, have been solved. In the A330P structure, the active site is constricted by the relocation of the Pro329 side chain into the substrate access channel, providing a basis for the distinctive C-H bond oxidation profiles given by the variant and the enhanced activity with small molecules. I401P, which is exceptionally active towards non-natural substrates, displays a number of structural similarities to substrate-bound forms of the wild-type enzyme, notably an off-axial water ligand, a drop in the proximal loop, and the positioning of two I-helix residues, Gly265 and His266, the reorientation of which prevents the formation of several intrahelical hydrogen bonds. Second-generation I401P variants gave high in vitro oxidation rates with non-natural substrates as varied as fluorene and propane, towards which the wild-type enzyme is essentially inactive. The substrate-free I401P haem domain had a reduction potential slightly more oxidising than the palmitate-bound wild-type haem domain, and a first electron transfer rate that was about 10 % faster. The electronic properties of A330P were, by contrast, similar to those of the substrate-free wild-type enzyme.

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“…human) P450 family members [3,4]. We could also extend its function beyond what was known to be catalyzed by P450s, making, for example, versions that hydroxylate gaseous alkanes (propane and ethane), transformations thought to lie in the functional realm of the methane monooxygenase family [5].…”

Section: Recent Research Contributions To Creating New Enzymesmentioning

confidence: 99%

Expanding the Enzyme Universe Through a Marriage of Chemistry and Evolution

Arnold¹

2014

New Chemistry and New Opportunities From the Expanding Protein Universe

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Present state of research on expanding enzyme catalysis beyond natureFor more than twenty years this laboratory has used directed evolution to modify enzymes. It is now widely accepted that directed evolution can change substrate specificity or reaction selectivity in desired ways, even if it sometimes remains difficult in practice. It is no longer surprising that enzymes adapt readily by accumulating beneficial mutations. And why should it be, since this is how nature tailors them for myriad biological roles? More difficult to grasp is how nature discovers new enzyme functions, particularly new catalytic activities. We know that the biological world's diverse catalytic repertoire is the product of evolution by natural selection, but we have little understanding of how nature's tinkering generates new functions. Sometimes we are (un)lucky enough to catch them in the act-e.g. the acquisition of antibiotic resistance or the ability to degrade man-made toxins. But for the vast majority of activities, the fossil record is nonexistent or too sparse to tell the molecular story. This leaves us without much guidance for evolving new enzymes in the laboratory. Consequently we are forced to contaminate our evolution experiments with knowledge-e.g. computational design [1,2]-in order to jumpstart the discovery process.If every bad catalyst could become a good one by directed evolution, part of the problem would be solved. We would be able to take any catalytic antibody or computationally-designed enzyme (or bovine serum albumin) and convert it into a great catalyst with multiple rounds of random mutagenesis and screening. We have learned the hard way, however, that not every bad catalyst lies at the base of a tall fitness peak, at least one that can be scaled by a random uphill walk. We therefore want to know what features make a protein with a new catalytic activity the potential mother of a whole new enzyme family. And, what are good ways to find new enzyme activities in the first place? Recent research contributions to creating new enzymesMy laboratory has been directing the evolution of a remarkable enzyme, a bacterial cytochrome P450, for at least a dozen years. This particular P450, from Bacillus megaterium, is quite specific-it catalyzes subterminal hydroxylation of fatty acids. As a family, however, the P450s are wonderfully diverse, catalyzing a wide range of reactions

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Engineered Alkane‐Hydroxylating Cytochrome P450_BM3 Exhibiting Nativelike Catalytic Properties

Cited by 225 publications

References 46 publications

Towards Preparative Scale Steroid Hydroxylation with Cytochrome P450 Monooxygenase CYP106A2

Towards Preparative Scale Steroid Hydroxylation with Cytochrome P450 Monooxygenase CYP106A2

Structural Basis for the Properties of Two Single‐Site Proline Mutants of CYP102A1 (P450_BM3)

Expanding the Enzyme Universe Through a Marriage of Chemistry and Evolution

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Engineered Alkane‐Hydroxylating Cytochrome P450BM3 Exhibiting Nativelike Catalytic Properties

Cited by 225 publications

References 46 publications

Towards Preparative Scale Steroid Hydroxylation with Cytochrome P450 Monooxygenase CYP106A2

Towards Preparative Scale Steroid Hydroxylation with Cytochrome P450 Monooxygenase CYP106A2

Structural Basis for the Properties of Two Single‐Site Proline Mutants of CYP102A1 (P450BM3)

Expanding the Enzyme Universe Through a Marriage of Chemistry and Evolution

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Engineered Alkane‐Hydroxylating Cytochrome P450_BM3 Exhibiting Nativelike Catalytic Properties

Structural Basis for the Properties of Two Single‐Site Proline Mutants of CYP102A1 (P450_BM3)