Altering the regioselectivity of the subterminal fatty acid hydroxylase P450 BM-3 towards γ- and δ-positions

doi:10.1016/j.jbiotec.2008.10.002

Journal of Biotechnology

Volume 139, Issue 1, 1 January 2009, Pages 115-117

https://doi.org/10.1016/j.jbiotec.2008.10.002 Get rights and content

Abstract

Cytochrome P450 BM-3 monooxygenase from Bacillus megaterium (CYP102A1) catalyzes the subterminal hydroxylation of fatty acids with a chain length of 12–22 carbons. Wild-type P450 BM-3 oxidizes saturated fatty acids at subterminal positions producing a mixture of ω-1, ω-2 and ω-3 hydroxylated products. Using a rational site-directed mutagenesis approach, three new elements have been introduced into the substrate binding pocket of the monooxygenase, which greatly changed the product pattern of lauric acid hydroxylation. Particularly, substitutions at positions S72, V78 and I263 had an effect on the enzyme regioselectivity. The P450 BM-3 mutants V78A F87A I263G and S72Y V78A F87A were able to oxidize lauric acid not only at δ-position (14% and 16%, respectively), but also produced γ- and β-hydroxylated products. δ-Hydroxy lauric and γ-hydroxy lauric acid are important synthons for the production of the corresponding lactones.

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Acknowledgments

Funding by Fachagentur Nachwachsende Rohstoffe and BMELV is gratefully acknowledged. TAD acknowledges funding by the German Academic Exchange service (DAAD) and DFG (SFB 706).

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Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins with broad chemical functionality that have widespread application as powerful biocatalysts given their high stereo- and regio-selectivity. A large variety of value-added compounds, including pharmaceuticals and natural products, can be synthesized by P450s, often with superior selectivity and at milder conditions than their chemical counterparts making them ideal alternative catalysts. This review addresses the most recent advances in P450-mediated syntheses and include enzyme engineering strategies that have expanded the stereo- and regio-selectivity of P450 steroid and fatty acid hydroxylation as well as facilitated C-N bond formation and cyclization, all of which have synthetic applications to produce value-added compounds. Approaches to overcome the limitations of P450 catalysis will also be discussed, including the use of decoy molecules to expand the substrate scope and enable the use of hydrogen peroxide, self-sufficient systems, cascade reactions and cofactor alternatives to improve efficiency and biotechnological viability.
Oxidization of benzo[a]pyrene by CYP102 in a novel PAHs-degrader Pontibacillus sp. HN14 with potential application in high salinity environment
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Benzo [a]pyrene (BaP) is a type of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs) with potent carcinogenicity; however, there are limited studies on its degradation mechanism. Here, a strain of Pontibacillus sp. HN14 with BaP degradation ability was isolated from mangrove sediments in Dongzhai Port, Hainan Province. Our study showed that biodegradation efficiencies reached 42.15% after Pontibacillus sp. HN14 was cultured with 20 mg L⁻¹ BaP as the sole carbon source for 25 days and still had degradability of BaP at a 25% high salinity level. Moreover, 9,10-dihydrobenzo [a]pyrene-7(8H)-one, an intermediate metabolite, was detected during BaP degradation in the HN14 strain. Genome analysis identified a gene encoding the CYP102(HN14) enzyme. The results showed that the E. coli strain with CYP102(HN14) overexpression could transfer BaP to 9,10-dihydrobenzo [a]pyrene-7(8H)-one with a conversion rate of 43.5%, indicating that CYP102(HN14) played an essential role in BaP degradation in Pontibacillus sp. HN14. Thus, our results provide a novel BaP biodegradation molecule, which could be used in BaP bioremediation in high salinity conditions. This study is the first to show that CYP102(HN14) had the BaP oxidization ability in bacteria. CYP102(HN14) could be essential in removing PAHs in saline-alkali soil and other high salt environments through enzyme immobilization.
Current state and future perspectives of cytochrome P450 enzymes for C–H and C=C oxygenation
2022, Synthetic and Systems Biotechnology
Cytochrome P450 enzymes (CYPs) catalyze a series of C–H and C=C oxygenation reactions, including hydroxylation, epoxidation, and ketonization. They are attractive biocatalysts because of their ability to selectively introduce oxygen into inert molecules under mild conditions. This review provides a comprehensive overview of the C–H and C=C oxygenation reactions catalyzed by CYPs and the various strategies for achieving higher selectivity and enzymatic activity. Furthermore, we discuss the application of C–H and C=C oxygenation catalyzed by CYPs to obtain the desired chemicals or pharmaceutical intermediates in practical production. The rapid development of protein engineering for CYPs provides excellent biocatalysts for selective C–H and C=C oxygenation reactions, thereby promoting the development of environmentally friendly and sustainable production processes.
Self-sufficient Cytochrome P450s and their potential applications in biotechnology
2021, Chinese Journal of Chemical Engineering
Cytochrome P450s (CYPs) are ubiquitously found in all kingdoms of life, playing important role in various biosynthetic pathways as well as degradative pathways; accordingly find applications in a vast variety of areas from organic synthesis and drug metabolite production to modification of biomaterials and bioremediation. Significantly, CYPs catalyze chemically challenging CH and CC activation reactions using a reactive high-valent iron-oxo intermediate generated upon dioxygen activation at their heme center, while the other oxygen atom is reduced to the level of water by electrons provided through a reductase partner protein. Self-sufficient CYPs, encoding their heme domain and reductase protein in a single polypeptide, facilitate increased catalytic efficiency and render a less complicated system to work with. The self-sufficient CYP enzyme from CYP102A family (CYP102A1, BM3) is among the earliest and most-investigated model enzymes for mechanistic and structural studies as well as for biotechnological applications. An increasing number of self-sufficient CYPs from the same CYP102 family and from other families have also been reported in last decade. In this review, we introduce chemistry and biology of CYPs, followed by an overview of the characteristics of self-sufficient CYPs and representative reactions. Enzyme engineering efforts leading to novel self-sufficient CYP variants that can catalyze synthetically useful natural and non-natural (nature-mimicking) reactions are highlighted. Lastly, the strategy and efforts that aim to circumvent the challenges for improved thermostability, regio- and enantioselectivity, and total turnover number; associated with practical use of self-sufficient CYPs are reviewed.
Characterization and modification of two self-sufficient CYP102 family enzymes from Bacillus amyloliquefaciens DSM 7 with distinct regioselectivity towards fatty acid hydroxylation
2021, Biochemical Engineering Journal
Two genes encoding for self-sufficient CYP102 family enzymes (bamf2522 and bamf0695) in Bacillus amyloliquefaciens DSM 7 have been cloned, purified, and subjected to biochemical characterization. Activity of both enzymes with various saturated and unsaturated fatty acid substrates were assessed. BAMF0695 mainly catalyzes hydroxylation at the ω-1, ω-2 and ω-3 positions of saturated linear fatty acids. In contrast, BAMF2522 exhibits broader product diversity by carrying out hydroxylation at the ω-1 to ω-7 positions of palmitic acid (C16:0), making it the only studied wild-type CYP102 enzyme that hydroxylates ω-6 and ω-7 positions for this substrate. Engineering of BAMF2522 for shifting hydroxylation towards in-chain positions and engineering of BAMF0695 for increasing hydroxylation towards ω-end have been performed. F89I mutant of BAMF2522 extends the product scope of palmitic acid from (ω-1) - (ω-7) to (ω-1) - (ω-9), with ω-7, ω-8 and ω-9 hydroxy fatty acids as the dominant products. On the other hand, F89I/A331V double mutant of BAMF0695 yields much higher ratio of ω-1 (63 %) product than the wild-type enzyme (32 %), indicating a significant shift of regioselectivity. Whereas BAMF2522 exhibited activity towards only myristoleic acid (C14:1) among the unsaturated fatty acids tested, BAMF0695 had a much broader substrate scope. Moreover, novel hydroxy products of arachidonic acid (C20:4) and myristoleic acid were obtained with the two wild-type enzymes.
Engineered P450 BM3 and cpADH5 coupled cascade reaction for β-oxo fatty acid methyl ester production in whole cells
2020, Enzyme and Microbial Technology
Citation Excerpt :
To determine the substrate and product spectrum of the generated biocatalyst co-expressing P450 BM3, cpADH5, and FhuA variants, fatty acid methyl ester substrates of different chain lengths (methyl butyrate, methyl valerate, methyl hexanoate, methyl heptanoate, and methyl octanoate) were tested. The P450 BM3 WT hydroxylates the subterminal carbon atom of the medium- to long-chain fatty acids and yields ω-1, ω-2 and ω-3 hydroxylated fatty acids [14,78]. Although distribution differs according to length and type of the substrate, ω-1 and ω-2 position favorably hydroxylated when saturated fatty acids tested as substrate [14].
Hydroxy- or ketone- functionalized fatty acid methyl esters (FAMEs) are important compounds for production of pharmaceuticals, vitamins, cosmetics or dietary supplements. Biocatalysis through enzymatic cascades has drawn attention to the efficient, sustainable, and greener synthetic processes. Furthermore, whole cell catalysts offer important advantages such as cofactor regeneration by cell metabolism, omission of protein purification steps and increased enzyme stability. Here, we report the first whole cell catalysis employing an engineered P450 BM3 variant and cpADH5 coupled cascade reaction for the biosynthesis of hydroxy- and keto-FAMEs. Firstly, P450 BM3 was engineered through the KnowVolution approach yielding P450 BM3 variant YE_M1_2, (R47S/Y51W/T235S/N239R/I401 M) which exhibited boosted performance toward methyl hexanoate. The initial oxidation rate of YE_M1_2 toward methyl hexanoate was determined to be 23-fold higher than the wild type enzyme and a 1.5-fold increase in methyl 3-hydroxyhexanoate production was obtained (YE_M1_2; 2.75 mM and WT; 1.8 mM). Subsequently, the whole cell catalyst for the synthesis of methyl 3-hydroxyhexanoate and methyl 3-oxohexanoate was constructed by combining the engineered P450 BM3 and cpADH5 variants in an artificial operon. A 2.06 mM total product formation was achieved by the whole cell catalyst including co-expressed channel protein, FhuA and co-solvent addition. Moreover, the generated whole cell biocatalyst also accepted methyl valerate, methyl heptanoate as well as methyl octanoate as substrates and yielded ω-1 ketones as the main product.

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Journal of Biotechnology

Abstract

Section snippets

Acknowledgments

References (19)

The case for open-source software in drug discovery

Drug Discov. Today

An active site substitution, F87V, converts cytochrome P450 BM-3 into a regio- and stereoselective (14S,15R)-arachidonic acid epoxygenase

J. Biol. Chem.

A single mutation in cytochrome P450 BM3 induces the conformational rearrangement seen upon substrate binding in the wild-type enzyme

J. Biol. Chem.

Omega-1, Omega-2 and Omega-3 hydroxylation of long-chain fatty acids, amides and alcohols by a soluble enzyme system from Bacillus megaterium

Biochim. Biophys. Acta

P450 BM3: the very model of a modern flavocytochrome

Trends Biochem. Sci.

Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium

J. Biol. Chem.

Identification and characterization of two functional domains in cytochrome P-450BM-3, a catalytically self-sufficient monooxygenase induced by barbiturates in Bacillus megaterium

J. Biol. Chem.

Rational re-design of the substrate binding site of flavocytochrome P450 BM3

FEBS Lett.

P450BM-3: absolute configuration of the primary metabolites of palmitic acid

Carcinogenesis

Cited by (68)

Harnessing heme chemistry: Recent advances in the biocatalytic applications of cytochrome P450 monooxgenases

Oxidization of benzo[a]pyrene by CYP102 in a novel PAHs-degrader Pontibacillus sp. HN14 with potential application in high salinity environment

Current state and future perspectives of cytochrome P450 enzymes for C–H and C=C oxygenation

Self-sufficient Cytochrome P450s and their potential applications in biotechnology

Characterization and modification of two self-sufficient CYP102 family enzymes from Bacillus amyloliquefaciens DSM 7 with distinct regioselectivity towards fatty acid hydroxylation

Engineered P450 BM3 and cpADH5 coupled cascade reaction for β-oxo fatty acid methyl ester production in whole cells