Page 115 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

5.1 Introduction 91

electron transfer from NADPH to the catalytic heme iron center of the P450.
In some cases, the second electron can be delivered from NADPH via cytochrome
b5 reductase and cytochrome b5 [21].
Bacterial P450s have been shown to interact with a much broader diversity of
redox partner proteins compared to their eukaryotic counterparts. These redox
partners of P450s are widely distributed in various combinations in nature and can
be utilized to classify P450 systems [18, 23].
While the number of newly annotated P450s is constantly increasing, the
identification of their natural redox partners (especially those associated to bacterial
P450s) is quite challenging because genes encoding P450s and their native redox
partners are not always located in neighboring loci. However, the application of
non-natural redox proteins from other P450 systems can successfully reconstitute
the activity of P450s [24–26].
In addition to the above-mentioned P450 systems, there are systems in which all
components are fused on a single polypeptide chain. Such P450 systems are consid-
ered to be self-sufficient, making them particularly attractive for biotechnological
applications because of their easier handling.
Cytochrome P450 proteins are ubiquitous in nature. Members of the P450
superfamily have been identified in viruses, bacteria, fungi, plants, insects, and ver-
tebrates [27]. Interestingly, not all prokaryotes possess P450 genes (e.g., Escherichia
coli has no CYP genes) and the number of all bacterial P450s identified so far is
considerably smaller than their eukaryotic counterparts.
P450s accept an extremely broad spectrum of organic substrate molecules,
including fatty acids, alkanes, alkenes, steroids, terpenes, polyaromatic hydro-
carbons, macrolides, and others. Of course, there is no single P450 capable of
accepting all these substrates. On the other hand, it is relatively common for a
group of P450s to oxidize a single substrate at different positions, or for a single
P450 to metabolize multiple substrates [28]. Moreover, some P450s are able to
mediate multiple sequential modifications on a single substrate.

5.1.3
General Overview of presented cascade types

In the following sections, we will describe cascade reactions of diverse types
involving P450s. According to Scheme 5.3, these include (1) multistep oxidations
catalyzed by a single P450, (2) multistep oxidations catalyzed by multiple P450s,
(3) cofactor regeneration cascades in P450 biocatalysis, and (4) implementation of
P450s in artificial enzyme cascades.
Reaction cascades of groups (1) and (2) will focus on physiologically occurring
P450 cascades and have not been yet explored for synthetic applications (or only
in very few parts). Cascades of groups (3) and (4) describe examples that have
been applied for synthetic purposes. Here, examples with isolated enzymes and
whole-cell approaches will be discussed.