Coenzyme Dependency

All type I BVMOs are strictly dependent on NADPH which delivers the electrons needed for catalysis. While the affinity for NADPH is high (KM 0.4-24 |imol L-1), type I BVMOs show no significant activity with NADH. The elucidation of the crystal structure of PAMO has revealed the molecular basis for this specificity. K336 appears to play a major role in binding of the 2'-phosphate of NADPH and is conserved in most type I BVMO sequences. Replacing this residue in HAPMO resulted in a drastically reduced affinity of NADPH while recognition of NADH improved [36]. The identification of residues recognizing the 2'-phosphate group enables structure-inspired enzyme engineering to create a BVMO accepting NADH. Such an enzyme mutagenesis approach will involve replacement of a number of residues, including K336. A BVMO which would be active with NADH is clearly advantageous for biocatalytic applications involving isolated enzyme as the non-phosphorylated cofactor is less expensive when compared with NADPH [47]. Engineering a BVMO which is active with NADH (and NADPH) might also prove to be valuable for conversions using whole cells as E. coli typically maintains high levels of NADH [48].

An alternative way to circumvent NADPH-dependent biocatalysis is to use artificial reducing agents. While for PAMO it has been shown that an artificial electron donor can be used [49], this approach has several significant disadvantages prohibiting applications. In general, artificial electron-donating catalysts/ agents are not as efficient as the natural coenzyme and often are costly. For PAMO it was also observed that binding of NADP+ is needed for efficient and enantiose-lective oxidations [49]. Electrochemical reduction has also been successfully applied to flavin-dependent monooxygenases [50]. Such an approach appears well suited for developing biosensors but is not (yet) applicable for synthetic purposes.

At the moment, the most effective method for in vitro regeneration of NAD(P) H is to couple the monooxygenase with an ancillary recycling enzyme. While for a long time glucose-6-phosphate dehydrogenase and formate dehydrogenase have been most popular as nicotinamide coenzyme recycling enzymes, several interesting alternative enzymes have been reported in the last years. Alcohol dehydro-genases from hyperthermophilic Archaea clearly are advantageous as recycling biocatalysts as they are (thermo)stable and tolerate organic solvents [47]. The use of an alcohol dehydrogenase in combination with a Baeyer-Villiger monooxygenase also makes it possible to perform a cascade reaction in which an alcohol is converted into the corresponding ester/lactone via formation of the ketone [51, 52].

Another attractive dehydrogenase for NAD(P)H recycling is phosphite dehydrogenase [53]. This enzyme is easily expressed in E. coli and is able to convert the inorganic substrate phosphite into phosphate while generating NADPH from NADP+. As the reaction is practically irreversible and phosphite can be bought as relatively inexpensive salt, this enzyme facilitates (cost-) efficient coenzyme regeneration.

Heal Yourself With Qi Gong

Heal Yourself With Qi Gong

Qigong also spelled Ch'i Kung is a potent system of healing and energy medicine from China. It's the art and science of utilizing breathing methods, gentle movement, and meditation to clean, fortify, and circulate the life energy qi.

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