Future Prospects

Recent years have seen a dramatic increase in our knowledge of the biodiversity of cytochrome P450 redox systems. Aside from those systems mentioned above, other recent examples demonstrating the flexibility of redox partner selection in microbial P450s include the presence of a P450-acyl CoA dehydrogenase fusion protein system in Pseudomonas fluorescens (Fig. 5.3f) and a P450 enzyme (CYP119A1) from the thermophilic bacterium Sulfolobus solfataricus whose activity is supported by a 7Fe ferredoxin (likely comprising both 3Fe-4S and 4Fe-4S clusters, in which the 3Fe-4S cluster is redox active in catalysis) and a 2-oxoacid (pyruvate)-dependent ferredoxin oxidoreductase (Fig. 5.3g) [133, 134]. The ther-mophilic system has the obvious advantage that costly nicotinamide coenzymes could be avoided through use of pyruvate as the electron donor [133].

In terms of the use of protein engineering to alter the catalytic properties of P450 systems, the most broad ranging and successful studies have been done with the P450cam and P450BM-3 systems (and BM-3's B. subtilis relatives CY-P102A2 and A3), producing variants capable of efficient hydroxylation of such molecules as alkanes, polyunsaturated fatty acids and drugs, and with potential applications in, for example, the generation of high value lipid mediators, ethanol and authentic human metabolites of drug molecules [e.g. 135, 136]. Despite notable breakthroughs, it is still the case that P450s are thought of as relatively fragile biocatalysts, vulnerable to inactivation through structural disruption and/ or cofactor dissociation. In particular, the P450s are known to undergo a conversion from an "active" P450 form (in which the heme Soret band is positioned close to 450 nm in the ferrous carbon monoxy complex) to an "inactive" P420 form (with the peak near 420 nm). However, recent biochemical and spectroscopic studies indicate that such transitions, at least for soluble P450s, likely reflect reversible protonation of the heme thiolate (to a thiol) and that substrate binding stabilizes the P450 form [102, 137, 138]. Further stabilization of heme binding may be achieved by covalent binding of the heme macrocycle to the protein, which occurs naturally in eukaryotic CYP4 enzymes by turnover-dependent linkage of a heme methyl group to a conserved acidic (glutamate) residue in the I helix [139, 140]. This was also shown to occur partially in a variant of P450cam in which a glutamate was engineered into the I helix at the appropriate position [141].

Other stability issues associated with extended turnover of P450 redox systems include the loss or degradation of non-covalently bound cofactors from the redox partners. In the BM-3-type (CYP102 class) P450-CPR fusion enzymes, for instance, FMN is relatively weakly bound by comparison with homologous flavo-doxin enzymes [142, 143]. Tightening binding of FMN in these enzymes is an obvious target for protein engineering studies, given the increasing interest in exploitation of the CYP102 (A1-A3) systems for production of chiral oxygenated molecules, and the requirement for extended turnover to facilitate high product yield. Many of the most biotechnologically attractive P450 systems are inactivated quite rapidly at temperatures above ~40 °C due to cofactor dissociation or irreversible structural change/aggregation [e.g. 143]. This has been addressed in theoretical, rational, and forced evolution studies of peroxygenase and other P450 systems, and also with reference to the known structural properties of P450s from thermostable organisms [e.g. 144, 145]. Clearly such studies may enable creation of more robust P450 catalysts that have greater useful lifetimes and higher thermostability.

A similar approach was used to produce modest increases in catalytic efficiency of the P450BM-3 system in the peroxide shunt reaction [121]. However, it appears highly unlikely that this reaction could ever compete with the efficiency of the "natural" NADPH-driven reaction, particularly given the fact that NADPH recycling systems can substantially diminish operating costs in this system and since peroxide-mediated heme destruction leads to oxidative degradation of the enzyme.

If progress in the next 50 years of P450 research is as great as in the first 50 years, then several further novel biocatalysts (likely catalytically efficient P450/ redox partner fusion enzymes) will be discovered and characterized, and protein engineering strategies to evolve activities and stabilize existing and novel enzymes will make these systems fully cost effective. This will allow the widespread exploitation of P450 oxygenases in biotechnological processes as efficient and safer alternatives to traditional organic synthesis methods.

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Heal Yourself With Qi Gong

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