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88 5 Multi-Enzyme Systems and Cascade Reactions Involving Cytochrome P450 Monooxygenases

is the possibility of a one-pot operation mode without the necessity of isolating
reaction intermediates, making such cascade reaction concepts highly efficient
and economically feasible.
The functionalization of nonactivated C–H bonds is crucial for the initial activa-
tion of simple (chemically inert) starting molecules. Hydroxylation of C–H bonds
directly leads to the formation of activated intermediates, which can further be
functionalized to high-value compounds widely needed as specialty chemicals and
for the development of active pharmaceutical ingredients. However, the function-
alization of nonactivated C–H bonds is one of the major challenges in chemistry.
Cytochrome P450 enzymes (P450s or CYPs) are capable of catalyzing such reactions
under mild reaction conditions. Although considerable progress has been achieved
regarding oxyfunctionalizations using chemical catalysts [5–7], P450s still remain
unsurpassed in their oxidizing ability and target specificity and are therefore highly
attractive catalytic tools for synthetic chemists and biotechnologists.
The use of P450s in artificial cascade reactions is a rather unexplored research
field, and corresponding reports are still very rare in the literature. However, their
ability to oxyfunctionalize cheap starting molecules to form valuable compounds,
combined with their broad natural functions as essential components in xenobiotics
degradation and biosynthesis of secondary metabolites, makes P450 enzymes
potential candidates for multi-enzyme processes.

5.1.2
Cytochrome P450 Monooxygenases

P450s form one of the largest enzyme superfamilies, with currently more than
21 000 annotated genes (http://drnelson.utmem.edu/cytochromeP450.html). Regard-
ing nomenclature, the root symbol CYP is followed by numbers representing the
particular families (whose members typically share more than 40% amino acid
identity), then by an alphabetical character for the respective subfamilies (whose
members share greater than 55% amino acid identity). Subfamilies are further sub-
divided into individual loci designated with a second set of numbers, for example,
CYP102A1, which represents the cytochrome P450 BM3 from Bacillus megaterium.
Trivial names of some well-characterized P450 enzymes are widely used in the
literature as well.
P450s belong to the class of oxidoreductases and are classically described as heme
b-containing monooxygenases that utilize the nicotinamide cofactors NADPH or
NADH to reductively cleave atmospheric dioxygen to form an oxyfunctionalized
product and a molecule of water. In many cases, the products represent hydroxylated
derivatives of the substrate at one of its carbon moieties, as demonstrated in the
generalized reaction mediated by these enzymes (Scheme 5.1).

RH NAD(P)H O 2 H + R OH NAD(P) + H O
2
Scheme 5.1 General hydroxylation reaction catalyzed by P450 monooxygenases. ‘‘RH’’ indi-
cates the substrate.