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108 5 Multi-Enzyme Systems and Cascade Reactions Involving Cytochrome P450 Monooxygenases

of CYP105A1. Co-crystallization of the wild-type enzyme and R84A mutant
with 1α,25-dihydroxyvitamin D3 combined with molecular docking and further
mutagenesis provided the structural basis for understanding the mechanism of
the two-step hydroxylation that activates vitamin D3 [76].

5.3
Artificial Cascade Reactions Involving P450s

5.3.1
Cascade Reactions Involving P450s and Cofactor Regenerating Enzymes

As mentioned in Section 5.1, almost all P450s require stoichiometric amounts
of pyridine cofactors for their activity. In order to render P450-catalyzed reactions
economically feasible, the cofactors must be regenerated. One of the most common
approaches for overcoming the stoichiometric need for the costly NAD(P)H cofactor
involves enzymatic regeneration systems that can be accomplished in vitro or in vivo
(in whole cells). Cofactor regeneration can drive the reaction to completion, which
further reduces the synthesis costs [77]. Different enzymatic cofactor systems have
been applied at a laboratory scale [78, 79].

5.3.1.1 Cofactor Regeneration in Cell-Free Systems (In Vitro)
Cofactors can be regenerated in vitro by various enzymes. In this section, we
discuss examples of P450 reactions using different enzymatic cofactor regener-
ation strategies. The cofactor regeneration enzymes include glucose-6-phosphate
dehydrogenase (G-6P-DH), alcohol dehydrogenase (ADH), formate dehydrogenase
(FDH), and others.

Glucose-6-Phosphate Dehydrogenase (G-6P-DH) Considering the complexity of
P450 redox chains, the simplest cascade reactions are based on the natural fusion
flavocytochrome CYP102A1 (P450 BM3), which is comprised of the N-terminal
P450 monooxygenase domain linked to the C-terminal diflavin reductase domain.
In this case, only one additional protein – a cofactor regenerating enzyme – is
needed. P450 BM3 is a fatty acid hydroxylase from B. megaterium that accepts
medium- to long-chain saturated and unsaturated fatty acids [80, 81].
The enzyme preferentially accepts NADPH, which can routinely be regenerated
by the application of G-6P-DH.
To our knowledge, the very first example for P450s in this regard describes
the use of G-6P-DH, which supported the hydroxylation of hydroxymyris-
tic acids to dihydroxymyristic acid products catalyzed by P450 BM3. The
+
reaction contained 10 mM substrate, 10 mM NADP , and 10 mM glucose-6-
phosphate along with P450 BM3 and a G-6P-DH. The reaction proceeded
regio- and stereoselectively at the ω-1 position of (R)-12-hydroxymyristic
acid and resulted in threo-12,13-dihydroxymyristic acid (80% (12S,13S)-12,13-
dihydroxymyristic acid and 20% (12S,13R)-12,13-dihydroxymyristic acid).