CYP102A P450 monooxygenases: comparative analysis and construction of cytochrome P450 chimera

The members of the CYP102A subfamily are in several ways unique. They are natural fusion proteins of approximately 117-119 kDa comprised of the N terminal monooxygenase domain and a FAD and FMN containing diflavin reductase domain. These fatty acid hydroxylases exhibit extraordinary activities of 3000-10000 min-1 in comparison to other P450 monooxygenases making them promising candidates for industrial applications. Up to now, cloning and characterisation of only three members of this subfamily have been published: CYP102A1 from Bacillus megaterium and CYP102A2 and CYP102A3 from Bacillus subtilis strain 168. We have currently cloned and characterised another member of this family, CYP102A7, and there are at least eight more genes for CYP102A monooxygenases, which have been identified in several genome sequencing projects. Multiple sequence alignment of all amino acid sequences revealed that the similarity between different members is between 50 and 90 % according to which these enzymes can be divided into four groups. The four cloned enzymes belonging to three different groups but with 65 to 70 % similarity were used for further investigation. Despite their appearance to be similar, they often exhibit different properties. For example, the new isolated CYP102A7 catalyses the deethylation of 7-ethoxycoumarin which is not accepted as substrate by CYP102A1, CYP102A2 and CYP102A3. The hydroxylation patterns produced by these four monooxygenases are also different. Additionally we investigated solvent and thermal stability of these P450 monooxygenases. For example, the monooxygenase domain of CYP102A1 is much more stable than those of CYP102A2and CYP102A3. On the other hand, its reductase domain is less stable than the corresponding ones of CYP102A2 and CYP102A3. Therefore we exchanged the natural more unstable reductase domain of CYP102A1 with the more stable reductase domain of CYP102A3, using the natural linker of CYP102A1. The new chimera was able to hydroxylate substrates within a wider temperature range (up to 50°C) compared to the parental enzymes, showed higher process stability and has a half-life at 50°C more than ten times longer than CYP102A1. Further we substituted the reductase domain of CYP102A1 by a thermostable analogue. In the genome of the thermophilic Geobacillus stearothermophilus a gene was found exhibiting 50 % protein similarity to the reductase domain of CYP102A1. The gene encodes a hypothetical alpha-subunit of a sulfite reductase, but also belongs to the diflavin reductases. This reductase was cloned downstream of the monooxygenase domain of CYP102A1. Activity of the foreign reductase within the chimera A1GR was confirmed by cytochrome c reduction. Next to the ability to transfer electrons to the heme iron, oxidation of C14-C18 fatty acids was proven. While the hydroxylation activity of CYP102A1 towards myristic and palmitic acid decreased to about 5 % of the initial rate after incubation at 49°C, the chimera exhibited no loss of activity under the same conditions.