Soluble Cytochromes P450

The unquestionable advantages of the use of recombinant bacteria such as Escherichia coli are of course rapid cell growth and (at least in principle) high expression levels, putting these organisms at the forefront of genetically modified organisms used in industry. Thus, bacteria are the obvious choice as expression systems for soluble cytochromes P450, even more so as these are generally of bacterial origin. Arguably, the most widely studied soluble P450 for biotechnolo-gical application nowadays is CYP102 from Bacillus megaterium because of its potential to hydroxylate a variety of alkanes, fatty acids, as well as aromatic compounds and also due to its high activity (cf. [54, 55]. Applying CYP102 in a whole cell system after its expression in E. coli does not require exogenous addition of the costly cofactor NADPH and appears to be a useful alternative to the enzymatic approach for the bioconversion of fatty acids [56]. Unfortunately, this enzyme is not a steroid hydroxylase, and indeed there are very few soluble P450s known to hydroxylate steroids (or seco-steroids) with sufficient regio- and stereospecificity to consider their use for biotechnological applications. The most prominent example is CYP106A2, the steroid 15ß-hydroxylase from Bacillus megaterium, which will therefore be discussed in more detail.

It has been known since 1958 that the B. megaterium strain ATCC 13368 is able to hydroxylate various steroid substrates in the 15ß-position [57], and since the 1970s Berg and co-workers have intensively investigated the hydroxylase system from this organism [58-63]. They were able to show that it consists of a cytochrome P450 enzyme (a 47.5 kDa protein later classified as CYP106A2), a strictly

NADPH-dependent FMN-containing protein (megaredoxin reductase), and an iron-sulfur protein (megaredoxin). The sequence of CYP106A2 shows high identity (63%) with CYP106, the P450BM-1 from B. megaterium ATCC 14581 [64]. The redox partners of CYP106A2 have not yet been cloned, but enzymatic activity can be obtained using the adrenal redox system consisting of adrenodoxin (Adx), and adrenodoxin reductase (AdR) [61, 65]. CYP106A2 is also able to interact with the electron transfer system from Bacillus subtilis [64] as well as with the etp1 protein from the fission yeast Schizosaccharomyces pombe [66] and with putidare-doxin reductase and putidaredoxin from Pseudomonasputida [67]. CYP106A2 was found to hydroxylate 3-oxo-A4-steroids at the 15P-position. While 3 P-hydroxy-A5-steroids are not converted by this enzyme [59, 60], the nature of the side-chain of the D-ring does not have an effect on the substrate-CYP106A2 interaction [64]. It is known that substrate binding to cytochromes P450 causes a high-spin shift of the heme iron resulting in a peak at around 390 nm and a trough at about 420 nm (cf. [6, 68]). However, spectroscopic studies of CYP106A2 have led to an unexpected result, as the spectrum of the substrate-free, oxidized form shows absorption maxima at 417 nm, 534 nm, and 566 nm. Interestingly, although the catalytic activity could readily be measured, the effect of substrate binding was not traceable by UV-vis spectroscopy. In contrast, an effect of substrate binding was detected using the CO stretch mode infrared spectrum indicating that deoxycorticosterone binds in the heme pocket near the iron ligand [65].

Berg et al. obtained a progesterone hydroxylation activity of 0.8 nmol 15P-hydroxyprogesterone per nmol CYP106A2 min-1 in an assay where they used the partially purified natural redox partners megaredoxin and megaredoxin reductase [59]. Later, a much higher production rate (337.3 ± 47.7 nmol per nmol CYP-106A2 min-1) could be accomplished by using an in vitro system consisting of Adx, AdR, and a NADPH-regenerating system [69].

In order to be able to engineer CYP106A2 for biotechnological purpose, knowledge of its 3D structure and an efficient high-level expression system are desirable. As the crystallization of the enzyme is still problematic, a high-quality homology model of the CYP106A2 structure was recently constructed in our group (Lisurek and Bernhardt, unpublished results). With respect to expression levels, preparations of native B. megaterium ATCC 13368 display merely 8 pmol CYP106A2 per mg protein [60]. Rauschenbach and co-workers therefore cloned the cDNA of CYP106A2 and expressed it in E. coli with a yield of 260 pmol CYP-106A2 per mg total protein [64]. By our group the expression yield could then be increased to 130 mg CYP106A2 per L culture (about 4400 pmol mg-1) [65]. Recently, further improvements by optimization of the expression conditions led to a yield of 8000 pmol P450 per mg total protein (Lisurek and Bernhardt, unpublished results). Over all, the amount of functional CYP106A2 could be increased by a factor of 1000 with respect to the level in the original bacterial strain. This certainly makes the recombinant system more applicable for the biotechnological production of steroids than the wild-type strain.

With all prerequisites for the genetic engineering of CYP106A2 by rational design being fulfilled, attempts were undertaken to change the selectivity of substrate hydroxylation catalyzed by CYP106A2. As mentioned above, the production of 11-hydroxylated steroids is of special interest for the pharmaceutical industry. Therefore, alignments of the structures of CYP106A2 and CYP11B1 were made to identify residues whose exchange might shift the regioselectivity of hydroxylation from the 15- to the 11-position. And indeed, such a shift could be shown by mutation of the amino acids at positions 395 and 397 in the CYP106A2 sequence. These mutations led to a significant increase (up to 4-fold) of 11a-OH progesterone production compared with the formation of 15P-OH progesterone. The effects on the other two known products of the CYP106A2 wild type, 9a- and 6P-OH progesterone, were less dramatic (Lisurek and Bernhardt, unpublished results).

Concomitant to the rational design described above, an attempt was also made to improve the properties of CYP106A2 by directed evolution. Usually, such an approach depends strongly on a fast screening system with a high accuracy. However, the main drawback in the work with steroids is, the lack of fast analytics. We therefore developed a fast and convenient system for the screening of improved steroid hydroxylation by CYP106A2 [70]. This system is based on a method described by Appel et al. [71] exploiting the observation that steroids exhibit fluorescence in an acidic environment.

To further simplify the screening procedure, a whole cell biocatalyst was developed [70]. Thus, mutants with improved activity can be screened in a rapid manner using the fluorescence technique. Moreover, medium-throughput screening using HPLC is also applicable using this whole cell system so that the same set of mutants can be used for studying changes in the activity as well as in the stereo- and regioselectivity of hydroxylation. Applying only one round of mutagene-sis by error-prone PCR and screening of the obtained mutants using the fluorescence assay, a CYP106A2 mutant with an approximately 3-fold increase of activity towards RSS could be obtained. Screening towards the substrate progesterone also revealed mutants with higher activity and, furthermore, changed the product pattern towards di- and polyhydroxylated progesterones.

Finally, by using a combination of rational design and the techniques of molecular evolution we were able to increase the amount of the 11a-hydroxylated product by a factor of 10 compared with the wild type [72]. These results show that the design of highly active bacterial steroid hydroxylases with designed regio-and stereoselectivity is in principle possible, although this goal is more difficult to reach than with other soluble P450s (like CYP101 and CYP102) that permit an easier detection of their reaction products (e.g. via color reactions).

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