Terminal Olefin (1-Alkene) Biosynthesis by a Novel P450 Fatty Acid Decarboxylase from Jeotgalicoccus Species (original) (raw)
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Journal of Biological Chemistry, 2014
Background: OleT JE oxidatively decarboxylates fatty acids to produce terminal alkenes. Results: OleT JE is an efficient peroxide-dependent lipid decarboxylase, with high affinity substrate binding and the capacity to be resolubilized from precipitate in an active form. Conclusion: OleT JE has key differences in active site structure and substrate binding/mechanistic properties from related CYP152 hydroxylases. Significance: OleT JE is an efficient and robust biocatalyst with applications in biofuel production.
Chemistry (Weinheim an der Bergstrasse, Germany), 2016
The cytochromes P450 are heme based monoxygenases or peroxygenases involved in vital reaction processes for human health. A recently described P450 peroxygenase, OleTJE, converts long chain fatty acids to terminal olefins and as such may have biotechnological relevance in biodiesel production. The reaction, however, produces significant amounts of α- and β-hydroxylation by-products and their origin are poorly understood. In this work we elucidate through a QM/MM study on the bifurcation pathways how the three possible products are generated and show how the enzyme can be further engineered for optimum desaturase activity. The studies show that the polarity and the solvent accessibility of the substrate in the binding pocket destabilize the OH rebound pathways and kinetically enable a thermodynamically otherwise unfavorable decarboxylation reaction. The origins of the bifurcation pathways are analyzed with valence bond models and highlight the differences in reaction mechanism.
Catalytic Determinants of Alkene Production by the Cytochrome P450 Peroxygenase OleTJE
The Journal of biological chemistry, 2017
The Jeotgalicoccus sp. peroxygenase cytochrome P450 OleTJE (CYP152L1) is a hydrogen peroxide-driven oxidase that catalyzes oxidative decarboxylation of fatty acids, producing terminal alkenes with applications as fine chemicals and biofuels. Understanding mechanisms that favor decarboxylation over fatty acid hydroxylation in OleTJE could enable protein engineering to improve catalysis or to introduce decarboxylation activity into P450s with different substrate preferences. In this manuscript, we have focused on OleTJE active site residues Phe79, His85 and Arg245 to interrogate their roles in substrate binding and catalytic activity. His85 is a potential proton donor to reactive iron-oxo species during substrate decarboxylation. The H85Q mutant substitutes a glutamine found in several peroxygenases that favor fatty acid hydroxylation. H85Q OleTJE still favors alkene production, suggesting alternative protonation mechanisms. However, the mutant undergoes only minor substrate binding-i...
Active Multi-Enzyme Assemblies for Long-Chain Olefinic Hydrocarbon Biosynthesis
Journal of bacteriology, 2017
Bacteria from different Phyla produce long-chain olefinic hydrocarbons derived from an OleA-catalyzed Claisen condensation of two fatty acyl-CoA substrates, followed by reduction and oxygen elimination reactions catalyzed by the proteins OleB, OleC, and OleD. In this report, OleA, OleB, OleC, and OleD were individually purified as soluble proteins, and all were found to be essential for reconstituting hydrocarbon biosynthesis. Recombinant co-expression of tagged OleABCD proteins from Xanthomonas campestris in Escherichia coli and purification over His6x- and FLAG-columns resulted in OleA separating whilst OleBCD purified together, irrespective of which of the four Ole proteins were tagged. Hydrocarbon biosynthetic activity of co-purified OleBCD assemblies could be reconstituted by adding separately-purified OleA. Immunoblots of non-denaturing gels using anti-OleC reacted with X. campestris crude protein lysate indicated the presence of a large protein assembly containing OleC in the...
FEBS letters, 2017
Jeotgalicoccus sp. 8456 OleTJE (CYP152L1) is a fatty acid decarboxylase cytochrome P450 that uses hydrogen peroxide (H2 O2 ) to catalyse production of terminal alkenes, which are industrially important chemicals with biofuel applications. We report enzyme fusion systems in which Streptomyces coelicolor alditol oxidase (AldO) is linked to OleTJE . AldO oxidizes polyols (including glycerol), generating H2 O2 as a co-product and facilitating its use for efficient OleTJE -dependent fatty acid decarboxylation. AldO activity is regulatable by polyol substrate titration, enabling control over H2 O2 supply to minimise oxidative inactivation of OleTJE and prolong activity for increased alkene production. We also use these fusion systems to generate novel products from secondary turnover of 2-OH and 3-OH myristic acid primary products, expanding the catalytic repertoire of OleTJE . This article is protected by copyright. All rights reserved.
The Highly Stereoselective Oxidation of Polyunsaturated Fatty Acids by Cytochrome P450BM-3
Journal of Biological Chemistry, 1996
Cytochrome P450BM-3 catalyzes NADPH-dependent metabolism of arachidonic acid to nearly enantiomerically pure 18(R)-hydroxyeicosatetraenoic acid and 14(S),15(R)-epoxyeicosatrienoic acid (80 and 20% of total products, respectively). P450BM-3 oxidizes arachidonic acid with a rate of 3.2 ؎ 0.4 mol/min/nmol at 30°C, the fastest ever reported for an NADPH-dependent, P450catalyzed reaction. Fatty acid, oxygen, and NADPH are utilized in an approximately 1:1:1 molar ratio, demonstrating efficient coupling of electron transport to monooxygenation. Eicosapentaenoic and eicosatrienoic acids, two arachidonic acid analogs that differ in the properties of the C-15-C-18 carbons, are also actively metabolized by P450BM-3 (1.4 ؎ 0.2 and 2.9 ؎ 0.1 mol/min/nmol at 30°C, respectively). While the 17,18-olefinic bond of eicosapentaenoic acid is epoxidized with nearly absolute regioand stereochemical selectivity to 17(S),18(R)-epoxyeicosatetraenoic acid (99% of total products, 97% optical purity), P450BM-3 is only moderately regioselective during hydroxylation of the eicosatrienoic acid-1,-2, and-3 sp 3 carbons, with 17-, 18-, and 19-hydroxyeicosatrienoic acid formed in a ratio of 2.4:2.2:1, respectively. Based on the above and on a model of arachidonic acid-bound P450BM-3, we propose: 1) the formation by P450BM-3 of a single oxidant species capable of olefinic bond epoxidation and sp 3 carbon hydroxylation and 2) that product chemistry and, thus, catalytic outcome are critically dependent on active site spatial coordinates responsible for substrate binding and productive orientation between heme-bound active oxygen and acceptor carbon bond(s). Miura and Fulco (1, 2) originally reported by that extracts of Bacillus megaterium could monooxygenate fatty acids. The enzyme responsible for this reaction was isolated, purified, cloned, sequenced, and found to be a 120-kDa fusion protein of a class II cytochrome P450 and NADPH-P450 reductase and was named cytochrome P450BM-3 (P450BM-3) 1 (3, 4).
Development of an orthogonal fatty acid biosynthesis system in E. coli for oleochemical production
Here we report recombinant expression and activity of several type I fatty acid synthases that can function in parallel with the native Escherichia coli fatty acid synthase. Corynebacterium glutamicum FAS1A was the most active in E. coli and this fatty acid synthase was leveraged to produce oleochemicals including fatty alcohols and methyl ketones. Coexpression of FAS1A with the ACP/CoA-reductase Maqu2220 from Marinobacter aquaeolei shifted the chain length distribution of fatty alcohols produced. Coexpression of FAS1A with FadM, FadB, and an acyl-CoA-oxidase from Micrococcus luteus resulted in the production of methyl ketones, although at a lower level than cells using the native FAS. This work, to our knowledge, is the first example of in vivo function of a heterologous fatty acid synthase in E. coli. Using FAS1 enzymes for oleochemical production have several potential advantages, and further optimization of this system could lead to strains with more efficient conversion to desired products. Finally, functional expression of these large enzyme complexes in E. coli will enable their study without culturing the native organisms.
The Biochemical journal, 2017
In the interest of decreasing dependence on fossil fuels, microbial hydrocarbon biosynthesis pathways are being studied for renewable, tailored production of specialty chemicals and biofuels. One candidate is long-chain olefin biosynthesis, a widespread bacterial pathway that produces waxy hydrocarbons. Found in three- and four-gene clusters, oleABCD encode the enzymes necessary to produce cis -olefins that differ by alkyl chain length, degree of unsaturation, and alkyl chain branching. The first enzyme in the pathway, OleA, catalyzes the Claisen condensation of two fatty acyl-coenzyme A molecules to form a β-keto acid. In this report, the mechanistic role of Xanthomonas campestris OleA Glu117 is investigated through mutant enzymes. Crystal structures were determined for each mutant as well as their complex with the inhibitor cerulenin. Complemented by substrate modelling, these structures suggest that Glu117 aids in substrate positioning for productive carbon-carbon bond formation....