Page 137 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

5.3 Artificial Cascade Reactions Involving P450s 113

NO 2
NO 2
O
P450 BM3 COO −
+ H
O 6
COO − O −
NADPH NADP +
6 p-Nitrophenolate ω-Oxydecanoic acid
10-pNCA
Transhydrogenase

NADH NAD +

Dihydroxyacetone Glycerol
GLD
Scheme 5.23 Coupling of P450 BM3-catalyzed 10-pNCA conversion with cofactor regenera-
tion system consisting of GLD and transhydrogenase.

putida relying on electron transfer from two proteins, called putidaredoxin (Pdx) and
NADH-dependent putidaredoxin reductase (PdR). P450 catalyzes the stereospe-
cam
cific oxidation of camphor to 5-exo-hydroxycamphor. The P450 electron transfer
cam
chain was investigated in various reaction systems, supporting NADH regeneration
in vitro. One example describes the reconstitution of P450 cam activity in microscale
aqueous compartments of stable water-in-oil emulsions formed with the nonionic
surfactant tetraethylene glycol dodecyl ether [94]. In this system, camphor hydroxy-
lation by the P450 system was combined with an enzymatic NADH regeneration
cam
system. Two different dehydrogenases, bacterial GLD and yeast ADH, were investi-
gated for NADH regeneration (Scheme 5.24). In the absence of dehydrogenases and
presence of 1 mM NADH, conversion of 2 mM camphor to 5-exo-hydroxycamphor
was only 5%. When one of the dehydrogenases was added to the system, a fivefold
increased conversion of camphor was achieved [94].

O P450cam, PdR, Pdx O
OH
5
Camphor 5-exo-Hydroxycamphor
NADH NAD +

Dihydroxyacetone Glycerol
or GLD or
or
acetaldehyde ADH ethanol
Scheme 5.24 P450 -catalyzed camphor oxidation using bacterial GLD or yeast ADH for
cam
cofactor regeneration.