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116 5 Multi-Enzyme Systems and Cascade Reactions Involving Cytochrome P450 Monooxygenases
O H O O
O 2 2
+ +
OH
O O
Progesterone 15β-Hydroxy-
(Prog) progesterone
(15β-Prog)
Adx CYP 106A2
AdR
NADPH + H + NADP +
ADH
E. coli
Acetone 2-Propanol
Figure 5.3 E. coli whole-cell oxidation of progesterone catalyzed by CYP106A2. Coexpressed
LbADH regenerates NADPH by oxidation of the cosubstrate 2-propanol. AdR and AdX are
the redox partner proteins for electron transfer to the P450. (Reproduced with permission
from [103]; (c) 2010, Wiley-VCH, Weinheim.)
mixtures of alcohol(s) and aldehyde/ketone. Moreover, most P450s catalyze only
the first hydroxylation step, yielding the corresponding alcohol. As a consequence,
a second enzyme is needed for the subsequent oxidation of alcohols to aldehydes
or ketones. Although this reaction concept is highly interesting, to date only a few
reports describe the implementation of a P450 with other enzymes, such as ADHs,
in artificial enzyme cascades. The next section will highlight the pioneering work
of different research groups in this particular scientific field.
5.3.3.1 Artificial Multi-Enzyme Cascades with Isolated Enzymes
Artificial cascades performed with isolated enzymes in vitro (cell-free extracts
or purified enzymes) provide several advantages compared to artificial cascades
in recombinant cells. Multi-enzyme reactions based on isolated enzymes can
easily be controlled in a desired manner, including key factors such as enzyme
combination in defined ratios and adjustment of specific reaction component ratios
(i.e., cofactors). This allows a more detailed control of the biocatalytic multi-enzyme
system in contrast to in vivo cascades where the above-mentioned factors are much
more difficult to control.
Two very recent examples of artificial in vitro cascade reactions combining a
regio- and chemoselective P450 with an ADH operating in a simultaneous one-pot
reaction mode have been reported by the groups of Gr¨ oger and Schwaneberg.
