Biotransformation by Bacterial P450 Enzymes

The monooxygenase activity of P450 enzymes requires two electrons from NAD(P)H to activate oxygen for substrate oxidation:

The two electrons are delivered to the P450 enzyme by electron transfer proteins which differ between organisms [6]. Most bacterial P450s utilize two electron transfer proteins, commonly a flavin-dependent reductase and an iron-sulfur fer-redoxin. Mammalian systems use a diflavin protein, NADPH-cytochrome P450 reductase, as a common electron transfer shuttle, while some bacterial systems such as the CYP102 family and P450RhF are self-sufficient proteins with fused electron transfer and P450 monooxygenase domains [6].

Biotransformation applications of P450 enzymes require a high level of enzyme expression, fast substrate oxidation rate, system stability for high total turnover, and high selectivity of product formation. Bacterial P450 monooxygenases are soluble, more stable than their eukaryotic counterparts, and exhibit higher catalytic activities and expression rates in recombinant hosts. These properties make them from a practical standpoint, promising candidates for biocatalysis. However, they often have narrow substrate specificity and only moderate selectivity. Protein engineering and directed evolution can be used to broaden the substrate range as well as to increase the turnover activity, coupling, and product selectivity. Another important factor is the yield of product based on NAD(P)H consumed, or the coupling efficiency. The enzyme/substrate match for non-natural substrates is often suboptimal and results in reducing equivalents from NAD(P)H being channelled away from product formation to give instead hydrogen peroxide and water. These "uncoupling" pathways have to be minimized during enzyme development.

The high cost of the NAD(P)H cofactor is a potential barrier to applying P450 enzymes in synthesis. A number of approaches to overcome this have been explored. Whole cell biotransformation faces the challenge of large reaction volume, slow mass transport, substrate and product toxicity, further oxidation or conversion of products by other cellular enzymes, and product recovery. The host organism can be immobilized to facilitate product recovery (e.g. in a flow reactor system), although mass transport will be slowed down further. Novel approaches have been developed to alter the property of the host cell wall to improve substrate uptake [7]. Two-phase reactions could minimize toxicity and product yields approaching 10 g L-1 have been achieved [8].

Applications in vitro require cofactor recycling; enzymatic regeneration has been successfully used for P450BM-3 utilizing D/L-isocitrate dehydrogenase [9], formate dehydrogenase [10], and alcohol dehydrogenase [11]. Other approaches are reviewed elsewhere in this volume (see Chapter 12). One attractive route that eliminates the need for cofactors is to supply electrons from an electrode [12], in particular if the enzyme is immobilized on the electrode to protect it from solvents and facilitate product recovery. However, this approach is still in early stage development. Turnover rates are slower than in solution and total turnover numbers are low. New avenues for delivering the electrons to fully functional enzymes at an electrode are required. The need for electrons could also be circumvented by the peroxide shunt in which alkyl peroxides and hydrogen peroxide or its precursors convert the ferric heme directly to the active ferryl intermediate. Enzyme inactivation and relatively slow reactions compared to the physiological activity are major obstacles. Directed evolution of P450BM-3 has generated mutants that are more active in the peroxide shunt and more stable to inactivation [13]. The bacterial enzymes, which work in an aqueous environment, are exposed to organ-ics during in vitro oxidations. Immobilizing the enzymes could reduce the denaturing effect of solvents [14]. The resistance of P450BM-3 to organic solvents such as DMSO has been increased by directed evolution [15].

Numerous expression systems have been applied for the heterologous expression of the P450cam and P450BM-3 in E. coli for whole cell biotransformation. Expression of the single polypeptide enzyme P450BM-3 is more straightforward: examples include pUC13 in E. coli JM103 [16], pGLW11 (pKK223-3 derivative with a tac promoter) in DH5a [17], pCWOri+ (tandem tac promoters) in a cata-lase-deficient strain [18], and pCYTEXP1, which contains the tandem promoters PR and PL, in DH5a [19]. In the pET28a+ construct, with a T7 promoter, P450BM-3 levels of up to 1200 nmol L-1 (-140 mg L-1) were reported [10]. This system is appropriate for expression both in microplate format and fed-batch fermentation for high-level enzyme production.

The first functional expression of the three-component P450cam system was in the form of a triple fusion (putidaredoxin reductase-putidaredoxin-P450cam) protein [20]. The activity of this in vivo system was lower than the in vitro reconstituted system because the linker peptides slowed down electron transfer. The pCWori+ vector was used to express the three proteins in a tricistronic system with each protein under the control of its own ribosome-binding site [21]. The expression level of the P450cam enzyme was lower than when it was expressed separately. Interestingly, when P450cam mutants were expressed in this system the host cells took on a blue coloration due to indigo formation via indole oxidation. The electron transfer proteins putidaredoxin reductase and putidaredoxin have been expressed from a pUC18 vector in the same host as the P450cam enzyme which was expressed from a pACYC177 derivative with the arabinose promoter to provide a system capable of oxidizing 1 g L-1 of camphor [22].

With increasing availability of metagenomes, genome sequences, and sophisticated heterologous expression methods, new P450 enzymes identified by bioin-formatics methods are readily expressed and isolated. These new enzymes will greatly expand the range of compounds that can be oxidized but equally important will be the different product selectivity to existing enzymes. However, even if substrates and their novel biotransformation were established, a significant difficulty facing the application of new enzymes is the low stability and activity of electron transfer chains to support the enzymatic activity. Many P450 genes are found without the associated electron transfer proteins immediately upstream or downstream, or nearby in the genome sequence. It then becomes necessary to screen many candidate genes to reconstitute the activity. Known electron transfer proteins from other P450 systems (e.g. P450cam, P450lin, and P450cin [6]) can also be tested. However, such "cross" reactivities are often low. Notably mammalian P450 activity can be supported by the human and yeast NADPH-cytochrome P450 reductase. This difficulty with bacterial systems makes the self-sufficient P450 systems such as the CYP102 family and P450RhF very attractive because the electron transport chains in these enzymes are fused to the P450 domain.

There is a real need for competent electron transfer chains for reconstituting the activity of the numerous other P450 enzymes that are "isolated" in the genome sequences but which display novel substrate specificity. The putidaredoxin reduc-tase-putidaredoxin system of P450cam [23], the fused flavin-ferredoxin domain of P450RhF [24], and a ferredoxin reductase-ferredoxin system from a Mycobacterium strain [25], have been used with some success. However, more of such electron transfer systems are needed to cover the diverse P450 enzymes being discovered.

Heal Yourself With Qi Gong

Heal Yourself With Qi Gong

Qigong also spelled Ch'i Kung is a potent system of healing and energy medicine from China. It's the art and science of utilizing breathing methods, gentle movement, and meditation to clean, fortify, and circulate the life energy qi.

Get My Free Ebook


Post a comment