Cofactor reconstitution

Figure 6.11 Examples of biohybrid catalytic systems, (a) Covalent coupling of a polystyrene tail to the enzyme CALB lipase the resulting biohybrid forms micellar fibers, (b) Cofactor reconstitution A polystyrene tail is connected to the horseradish peroxidase enzyme via the cofactor ferri-protoporphyrin IX. the resulting...

Hamachi I, Tanaka S,Tsukiji S, Shinkai S, Oishi S. Design and semisynthesis of photoactive myoglobin bearing ruthenium tris(2,2 -bipyridine) using cofactor-reconstitution. Inorg Chem 1998 37 4380-8. 

Hamachi I, Shinkai S. Chemical modification of the structures and functions of proteins by the cofactor reconstitution method. Eur I Org Chem 1999 539 19. 

Boerakker MJ, Hannink JM, Bomans PH, Fredeiik PM, Nolle RJ, Meijer EM, Sommerdijk NA (2002) Giant amphiphiles by cofactor reconstitution. Angew Chem Int Ed 41 4239-4241... 

FIGURE 6-9 Electrical contact of a flavoenzyme by its reconstitution with a relay-FAD semisynthetic cofactor. Fc = ferrocene. (Reproduced with permission from reference 2.)... 

The molybdenum cofactor was liberated from D. gigas AOR, and under appropriate conditions was transferred quantitatively to nitrate reductase in extracts of Neurospora crassa nit-1 mutant) to yield active nitrate reductase 217). On the basis of molybdenum content, the activity observed for reconstitution with molybdenum cofactor of D. gigas was lower (25%) than the values observed for the procedure using extractable molybdenum cofactor of XO, used as reference. This result can now be put in the context of the difference in pterin present (MPT-XO and MCD-AOR) 218). 

Moreover, an electron transfer chain could be reconstituted in vitro that is able to oxidize aldehydes to carboxylic acids with concomitant reduction of protons and net production of dihydrogen (213, 243). The first enzyme in this chain is an aldehyde oxidoreductase (AOR), a homodimer (100 kDa) containing one Mo cofactor (MOD) and two [2Fe—2S] centers per subunit (199). The enzyme catalytic cycle can be regenerated by transferring electrons to flavodoxin, an FMN-con-taining protein of 16 kDa (and afterwards to a multiheme cytochrome and then to hydrogenase) ... 

Saito et al. (134) found that the cytosolic nitroreductase activity was due to DT-diaphorase, aldehyde oxidase, xanthine oxidase plus other unidentified nitroreductases. As anticipated, the microsomal reduction of 1-nitropyrene was inhibited by 0 and stimulated by FMN which was attributed to this cofactor acting as an electron shuttle between NADPH-cytochrome P-450 reductase and cytochrome P-450. Carbon monoxide and type II cytochrome P-450 inhibitors decreased the rate of nitroreduction which was consistent with the involvement of cytochrome P-450. Induction of cytochromes P-450 increased rates of 1-aminopyrene formation and nitroreduction was demonstrated in a reconstituted cytochrome P-450 system, with isozyme P-448-IId catalyzing the reduction most efficiently. 

FIGURE 33 Electrical contacting of a flavoenzyme by its reconstitution with a relay-FAD semisynthetic cofactor. 

Stallmeyer, B., Schwarz, G., Schulze, J., Nerlich, A., Reiss, J., Kirsch, j., and Mendel, R. R. The neurotransmitter receptor-anchoring protein gephyrin reconstitutes molybdenum cofactor biosynthesis in bacteria, plants, and mammalian cells, Proc Natl Acad Sci USA 1999, 96, 1333-1338. 

The reconstitution of apo-enzymes on relay-cofactor monolayer-functionalized electrodes was used to align redox enzymes and to establish electrical contact... 

Figure 12.2 (a) Reconstitution of an apo-enzyme on a relay-cofactor monolayer for the... 

Au NPs (1.2 nm) that include a single /V-hydroxysuccinimide-active ester functionality were modified with 2-amino-ethyl-flavin adenine dinucleotide, (5), and apo-glucose oxidase was reconstituted on the FAD cofactor units to yield the Au NP-GOx hybrid (Fig. 12.6a). The resulting hybrids were linked to the Au surface by different dithiol bridging units (8), (9), and (10). The resulting NP-functionalized glucose oxidase, GOx, exhibited electrical contact with the electrode surface, and the Au NPs... 

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