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112 5 Multi-Enzyme Systems and Cascade Reactions Involving Cytochrome P450 Monooxygenases

The same FDH was utilized for the regeneration of NADPH consumed by P450
BM3-catalyzed reactions in biphasic systems with octane and cyclohexane. In the
+
presence of organic solvents, the NADP -dependent FDH mutant demonstrated
high operational stability under almost all tested reaction conditions [89].

Other Enzymatic Cofactor Regeneration Strategies Although the described systems
work reasonably well, further reduction of costs for enzymatic oxyfunctionalizations
can be achieved by engineering NADPH-dependent P450s to accept NADH [90, 91].
NADH has the advantage that it is about 10 times less expensive and more stable
+
than NADPH. Furthermore, more NAD -dependent enzymes at lower prices are
available for cofactor regeneration. For instance, using homology modeling and
site-directed mutagenesis, P450 BM3 mutants have been constructed showing
altered cofactor specificity from NADPH to NADH. The best mutant possessing
the W1046S/R966D had comparable affinity to both NADH and NADPH and
displayed a more than 2× higher activity when NADH was used [89].
Another strategy for NADPH regeneration involves the switching of the cofac-
+
tor specificity of the regenerating enzyme from NAD + to NADP . Structural
details building a rational basis for switching cofactor specificity were reported for
+
the NAD -dependent FDH from Candida boidinii. By combining structure-based
analysis, multiple-sequence alignment, and saturation mutagenesis, the double
7
mutant D195Q/Y196H was engineered, which displayed more than 2 × 10 -fold
+
improvement in overall catalytic efficiency with NADP . This mutant was tested
for NADPH regeneration during P450-catalyzed conversions with CYP102A2 from
Bacillus subtilis (a homolog of P450 BM3). Using a 1250-fold excess of 12-pNCA
+
over NADP , 20% substrate conversion and a total turnover number (TTN) of 300
were achieved after 40 min [92].
A P450 BM3-catalyzed reaction was also linked to a two-step cofactor regeneration
approach in a cell-free system. The two-step cofactor regeneration of redox cofac-
+
tors, NADH and NADPH, was constructed by NAD -dependent bacterial glycerol
dehydrogenase (GLD) and bacterial soluble transhydrogenase (both from E. coli).
+
P450 BM3 oxidized NADPH to NADP upon concomitant hydroxylation of the
model substrate 10-pNCA. In the developed multi-enzyme system, NADH was pro-
duced by GLD using glycerol as substrate. Hydrides were subsequently transferred
from NADH to NADP + by transhydrogenase (Scheme 5.23). The comparison
of this two-enzyme cofactor regeneration system with NADPH regeneration by
glucose dehydrogenase (GDH) demonstrated that transhydrogenation catalyzed by
transhydrogenase was the rate-determining step. Nevertheless, whereas only 34%
of 50 μM pNCA was converted when supported by 50 μM NADPH, with the two-step
cofactor regeneration the same amount of substrate was completely converted after
+
+
adding 5 μMNAD and NADP [93].
Complex Multicomponent Cofactor Regenerating Systems More complex multi-
component cofactor regeneration systems have been described for nonfused P450
enzymes, which are accompanied by electron transfer between independent redox
partners. The best characterized system in this respect is P450 from Pseudomonas
cam