Laboratory evolution of P450 BM-3 for mediated electron transfer.
ABSTRACT Preparative synthesis with P450 monooxygenases is hampered in cell-free systems by the requirement for cofactors such as NAD(P)H as reduction equivalents. A validated medium-throughput screening system was designed for improving P450 monooxygenases by mediated electron transfer with zinc/cobalt(III)sepulchrate (Zn/Co(III)sep) as an alternative and cost-effective cofactor system. The monooxygenase P450 BM-3 F87A was used as a model system for developing the screening system in a 96-well format. A coefficient of variation of less than 10% was achieved under optimized screening conditions. The mediator evolution screen was validated by comparing the activity of P450 BM-3 to P450 BM-3 F87A and by screening a saturation mutagenesis library at amino acid position R47. For mediated electron transfer, two double mutants P450 BM-3(F87A R47F) and P450 BM-3 (F87A R47Y) were identified with a two-threefold increased catalytic efficiency (up to 32 microM(-1) min(-1) for P450 BM-3(F87A R47F) and 34 microM(-1) min(-1) for P450 BM-3 (F87A R47Y)) compared to P450 BM-3 F87A. The kinetic constants of the double mutants are, in contrast to those of P450 BM-3 F87A, dependent on Co(III)sep concentration in the presence of NADPH. kcat increases from 145 min(-1) (0.25 mM Co(III)sep) to 197 min(-1) (0.5 mM Co(III)sep), and Km decreases simultaneously from 7.0 microM to 3.7 microM, for P450 BM-3 (F87A R47F). For P450 BM-3 (F87A R47Y), kcat increases from 138 min(-1) (0.25 mM Co(III)sep) up to 187 min(-1) (0.5 mM Co(III)sep), and Km decreases from 8.2 microM to 4.2 microM. Due to lower Km values, the catalytic efficiencies were improved six times for P450 BM-3 (F87A R47F) and three times for P450 BM-3 (F87A R47Y), when comparing catalytic efficiencies of the mediated electron-transfer system to the natural reduction equivalent NADPH.
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ABSTRACT: We have converted cytochrome P450 BM-3 from Bacillus megaterium (P450 BM-3), a medium-chain (C12-C18) fatty acid monooxygenase, into a highly efficient catalyst for the conversion of alkanes to alcohols. The evolved P450 BM-3 exhibits higher turnover rates than any reported biocatalyst for the selective oxidation of hydrocarbons of small to medium chain length (C3-C8). Unlike naturally occurring alkane hydroxylases, the best known of which are the large complexes of methane monooxygenase (MMO) and membrane-associated non-heme iron alkane monooxygenase (AlkB), the evolved enzyme is monomeric, soluble, and requires no additional proteins for catalysis. The evolved alkane hydroxylase was found to be even more active on fatty acids than wild-type BM-3, which was already one of the most efficient fatty acid monooxgenases known. A broad range of substrates including the gaseous alkane propane induces the low to high spin shift that activates the enzyme. This catalyst for alkane hydroxylation at room temperature opens new opportunities for clean, selective hydrocarbon activation for chemical synthesis and bioremediation.Nature Biotechnology 12/2002; 20(11):1135-9. · 32.44 Impact Factor
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ABSTRACT: A unique cytochrome P-450-dependent fatty acid monooxygenase from Bacillus megaterium ATCC 14581 is strongly induced by phenobarbital (Narhi, L. O., and Fulco, A. J. (1982) J. Biol. Chem. 257, 2147-2150) and many other barbiturates (Kim, B.-H., and Fulco, A. J. (1983) Biochem. Biophys. Res. Commun. 116, 843-850). This monooxygenase has now been purified to homogeneity from pentobarbital-induced bacteria as a single polypeptide with a molecular weight of 119,000 +/- 5,000 daltons. In the presence of NADPH and O2, it can catalyze the oxygenation of long chain fatty acids without the aid of any other protein. The enzyme has a catalytic center activity of 4,600 nmol of fatty acid oxygenated per nmol of P-450 (the highest activity yet reported for a P-450-dependent monooxygenase) and also functions as a highly active cytochrome c reductase in the presence of NADPH. The purified holoenzyme is a soluble protein containing 40 mol % hydrophobic amino acid residues and 1 mol each of FAD and FMN/mol of heme. It is isolated and purified in the low spin form but is converted to the high spin form in the presence of long chain fatty acids. The enzyme, which catalyzes the omega-2 hydroxylation of saturated fatty acids and the hydroxylation and epoxidation of unsaturated fatty acids has its highest affinity (Km = 2 +/- 1 microM) for the C15 and C16 chain lengths.Journal of Biological Chemistry 07/1986; 261(16):7160-9. · 4.65 Impact Factor
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ABSTRACT: In a previous publication (Narhi, L. O. and Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169) we described the characterization of a soluble 119,000-dalton P-450 cytochrome (P-450BM-3) that was induced by barbiturates in Bacillus megaterium. This single polypeptide contained 1 mol each of FAD and FMN/mol of heme and, in the presence of NADPH and O2, catalyzed the oxygenation of long-chain fatty acids without the aid of any other protein. We have now utilized limited trypsin proteolysis in the presence of substrate to cleave P-450BM-3 into two polypeptides (domains) of about 66,000 and 55,000 daltons. The 66-kDa domain contains both FAD and FMN but no heme, reduces cytochrome c in the presence of NADPH, and is derived from the C-terminal portion of P-450BM-3. The 55-kDa domain is actually a mixture of three discrete peptides (T-I, T-II, and T-III) separable by high performance liquid chromatography. All three contain heme and show a P-450 absorption peak in the presence of CO and dithionite. The major component, T-I (Mr = 55 kDa), binds fatty acid substrate and has an N-terminal amino acid sequence identical to that of intact P-450BM-3, an indication that this domain constitutes the N-terminal portion of the 119-kDa protein. T-II (54 kDa) is the same as T-I except that it is missing the first nine N-terminal amino acids and does not bind substrate. T-III (Mr = 53.5 kDa) has lost the first 15 N-terminal residues and does not bind substrate. Since trypsin digestion of P-450BM-3 carried out in the absence of substrate yields T-II and T-III but no T-I, it appears that 1 or more residues of the first nine N-terminal amino acids of this protein are intimately involved in substrate binding. Although both the heme- and flavin-containing tryptic peptides retain their original half-reactions, fatty acid monooxygenase activity cannot be reconstituted after proteolysis, and the two domains, once separated, show no affinity for each other. In most respects, the reductase domain of P-450BM-3 more closely resembles the mammalian microsomal P-450 reductases than it does any known bacterial protein.Journal of Biological Chemistry 06/1987; 262(14):6683-90. · 4.65 Impact Factor