Altering the regioselectivity of the subterminal fatty acid hydroxylase P450 BM-3 towards gamma- and delta-positions.

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 omega-1, omega-2 and omega-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 delta-position (14% and 16%, respectively), but also produced gamma- and beta-hydroxylated products. delta-Hydroxy lauric and gamma-hydroxy lauric acid are important synthons for the production of the corresponding lactones.

Identification and functional expression of a Delta9-fatty acid desaturase from Psychrobacter urativorans in Escherichia coli.

The Delta9-fatty acid desaturase is a key enzyme in the synthesis of unsaturated fatty acids. The fatty acid composition of membrane phospholipids in Psychrobacter urativorans is characterized by a high degree of desaturation at Delta9 position. Based on CODEHOP-mediated PCR strategy, a novel gene designated as PuFAD9, putatively encoding a Delta9-fatty acid desaturase (PuFAD9), was isolated from P. urativorans. The gene consists of 1,455 bp and codes for 484 amino acids. Analysis of the amino acid sequence reveals three histidine clusters and a hydropathy profile, typical for membrane-bound desaturases. Activity of the PuFAD9 protein, recombinantly expressed in Escherichia coli was confirmed by GC-MS analysis of the cellular fatty acid composition. It was found that the ratio between palmitoleic and palmitic acid in E. coli cells heterologously expressing the PuFAD9 gene was significantly affected by IPTG induction and the growth temperature.

Cloning, expression and characterisation of CYP102A7, a self-sufficient P450 monooxygenase from Bacillus licheniformis.

Cytochrome P450 monooxygenases of the CYP102A subfamily are single-component natural fusion proteins consisting of a heme domain and a diflavin reductase. The characterised CYP102A enzymes are fatty acid hydroxylases with turnover rates of several thousands per minute. In search of new P450s with similar activities, but with a broader substrate spectrum, we cloned, expressed and characterised CYP102A7 from Bacillus licheniformis. As expected, CYP102A7 was active towards medium-chain fatty acids but showed a strong preference for saturated over unsaturated fatty acids, which could not be observed for either of the CYP102A members so far. Besides fatty acids, CYP102A7 was able to catalyse the oxidation of cyclic and acyclic terpenes with high activity and coupling efficiency. For example, (R)-(+)-limonene was converted with activity of 220 nmol nmol P450(-1) min(-1) and 80% coupling. Unusual for enzymes of the CYP102A subfamily was the deethylation activity of CYP102A7 towards 7-ethoxycoumarin. Furthermore, this monooxygenase, though having a moderate thermal stability, exhibited 50% of its initial activity in the presence of 26% DMSO. Comparison of the homology models of CYP102A7 and other members of the CYP102A subfamily revealed distinct differences in the shape of the substrate access channel and the active site, which might explain differences in catalytic properties of these homologous enzymes.

Cytochrome P450 monooxygenase from Clostridium acetobutylicum: a new alpha-fatty acid hydroxylase.

Cytochrome P450 monooxygenase from the anaerobic microorganism Clostridium acetobutylicum (CYP152A2) has been produced in Escherichia coli. CYP152A2 was shown to bind a broad range of saturated and unsaturated fatty acids and corresponding methyl esters and demonstrated a high peroxygenase activity of up to 200min(-1) with myristic acid. Although a high concentration of hydrogen peroxide of 200microM was necessary for high activities of the enzyme, it led to a fast enzyme inactivation within 2-4min. This might reflect the natural function of CYP152A2 as a rapid hydrogen peroxide scavenging enzyme. In two different reconstituted systems with NADPH, CYP152A2 was able to convert 10 times more substrate, if provided with flavodoxin and flavodoxin reductase from E. coli and even 30-40 times more substrate with the CYP102A1-reductase from Bacillus megaterium. According to the clear preference for hydroxylation at alpha-position, CYP152A2 can be referred to as fatty acid alpha-hydroxylase.

Recombinant production of human microsomal cytochrome P450 2D6 in the methylotrophic yeast Pichia pastoris.

Microsomal cytochrome P450 monooxygenases of groups 1-3 are mainly expressed in the liver and play a crucial role in phase 1 reactions of xenobiotic metabolism. The cDNAs encoding human CYP2D6 and human NADPH-P450 oxidoreductase (CPR) were transformed into the methylotrophic yeast Pichia pastoris and expressed with control of the methanol-inducible AOX1 promoter. The determined molecular weights of the recombinant CYP2D6 and CPR closely matched the calculated values of 55.8 and 76.6 kDa. CPR activity was detected by conversion of cytochrome c by using isolated microsomes. Nearly all of the recombinant CYP was composed of the active holoenzyme, as confirmed by reduced CO difference spectra, which showed a single peak at 450 nm. Only by coexpression of human CPR and CYP was CYP2D6 activity obtained. Microsomes containing human CPR and CYP2D6 converted different substrates, such as 3-cyano-7-ethoxycoumarin, parathion and dextrometorphan. The kinetic parameters of dextrometorphan conversion closely matched those of CYP2D6 from other recombinant expression systems and human microsomes. The endogenous NADPH-P450 oxidoreductase of Pichia pastoris seems to be incompatible with human CYP2D6, as expression of CYP2D6 without human CPR did not result in any CYP activity. These recombinant strains provide a novel, easy-to-handle and cheap source for the biochemical characterisation of single microsomal cytochromes, as well as their allelic variants.