FAD cofactor

Figure 6 Model structure of CHMO. The FAD cofactor is shown as sticks. Mutations observed in a directed evolution study resulting in altered enantioselectivities are highlighted (spheres). " Note that a number of mutations were found in double/triple mutants and cannot be linked with certainty to the altered catalytic properties. Mutations in double/triple mutants that are on the...

The catalytic activity of MAO can be simply characterized as two half-reactions (Fig. 4.33). In the first half-reaction, the amine substrate is oxidized and the FAD cofactor is reduced. In the second half-reaction, the imine product is released and the FAD cofactor reoxidized generating peroxide. The released imine chemically hydrolyzes to the corresponding aldehyde. 

In order to maintain adequate levels of GSH, the oxidized dimer is then reduced back to the original thiol components. This is achieved using the enzyme GSH reductase in a reaction involving NADPH and FAD cofactors (see Section 15.1.1). 

Figure 2.7 Structures of a lysine-specific hystone demethylase 1 (LSDl). In all three pictures, the LSDl protein is in the same orientation, (a) The human LSDl structure (PDB code 2HK0) with the tower domain (top two helices), just below the AOL domain, (harboring the FAD cofactor and the substrate-binding subdomain).The protein is represented schematically, with the helices, p-strands and coil regions colored in red, yellow and green, respectively. The FAD cofactor is in stick representation with the carbon, oxygen, phosphorus and nitrogen...

A single electron transfer (SET) mechanism involving initial transfer of an electron from the FAD cofactor has been proposed for MAO-catalyzed oxidations [10]. An alternative polar nucleophilic mechanism has also received support [11]. 

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... 

The enzyme p-hydroxybenzoate hydroxylase utilizes a cosubstrate together with 02 to form 3,4-dihydroxybenzoate. Indicate the mechanisms by which the bound FAD cofactor participates in the reaction. 

In the case of MAO, oxidation of the substrates is coupled to the reduction of flavin adenine dinucleotide (FAD) cofactor. The product of the reaction is the amine of the substrate, which hydrolyzes spontaneously to yield the corresponding aldehyde and ammonia. Reoxidization of the cofactor by molecular oxygen produces H202, according to the following reactions ... 

Zayats M, Katz E, Willner I. Electrical contacting of glucose oxidase by surface-reconstitution of the apo-protein on a relay-boronic acid-FAD cofactor. Journal of the American Chemical Society 2002, 124, 2120-2121. 

To optimize the photoswitchable bioelectrocatalytic features of the protein, site-specific functionalization or mutation of the active site microenvironment is essential. This was accomplished by a semisynthetic approach involving the reconstitution of the flavoenzyme-glucose oxidase with a semisynthetic photoisomerizable FAD cofactor (Scheme 9).1511 The photoisomerizable nitrospiropyran carboxylic acid (24) was covalently coupled to N6-(2-aminoethyl)-FAD (25), to yield the synthetic photoisomerizable nitrospiropyran-FAD cofactor 26a (Scheme 9(A)). The native FAD cofactor was removed from glucose oxidase, and the synthetic photoisomeriz-able-FAD cofactor 26a was reconstituted into the apo-glucose oxidase (apo-GOx), to yield the photoisomerizable enzyme 27a (Scheme 9(B)). This reconstituted protein... 

A further approach to controlling electrical communication between redox proteins and their electrode support through a photo-command interface includes photo stimulated electrostatic control over the electrical contact between the redox enzyme and the electrode in the presence of a diffusional electron mediator (Scheme 12).[58] A mixed monolayer, consisting of the photoisomerizable thiolated nitrospiropyran units 30 and the semi-synthetic FAD cofactor 25, was assembled on an Au electrode. Apo-glucose oxidase was reconstituted onto the surface FAD sites to yield an aligned enzyme-layered electrode. The surface-reconstituted enzyme (2 x 10-12 mole cm-2) by itself lacked electrical communication with the electrode. In the presence of the positively charged, protonated diffusional electron mediator l-[l-(dimethylamino)ethyl]ferrocene 29, however, the bioelectrocatalytic functions of the enzyme-layered electrode could be activated and controlled by the photoisomerizable component co-immobilized in the monolayer assembly (Figure 12). In the... 

Scheme 12 Surface reconstitution of apo-glucose oxidase on a mixed monolayer associated with an electrode consisting of an FAD cofactor and photoisomerizable nitrospiropyran units, and reversible photoswitching of the bioelectrocatalytic functions of the enzyme electrode.

Brown, E.D., and J.M. Wood (1993). Conformational change and membrane association of the PutA protein are coincident with reduction of its FAD cofactor for proline. J. Biol. Chem. 268 8972-8979. 

Figure 12.4 Photoinduced bioelectrocatalyzed oxidation of glucose to gluconic acid by glucose oxidase (COD) reconstituted with a nitrospiropyran-modified FAD cofactor (Sp-FAD) assembled as a monolayer on the Au electrode. Fhe Sp-FAD reveals reversible photoisomeriz-able properties yielding nitromerocyanine-FAD isomer (MRH+-FAD) [34]...

Willner I, Blonder R, Katz E. Reconstitution of apo-glucose oxidase with a nitrospiro-pyran-modified FAD cofactor yields a photoswitchable biocatalysts for amperometric transduction of recorded optical signals. J Am Chem Soc 1996 118 5310-11. 

PHBH is the protype of the flavoprotein aromatic hydroxylases. Each subunit of this dimeric enzyme contains two active sites which, during catalysis, are alternately visited by the isoalloxazine ring of the FAD cofactor (31). Catalysis is iiutiated by reduction of the flavin in the exterior active site. The reduced flavin then moves to the interior active site where the reactions with oxygen occur. A similar conformational flexibility of the FAD cofactor has been observed in the crystal structures of phenol hydroxylase (EC 1.14.13.7) and 3-hydroxybenzoate 4-hydroxylase (EC 1.14.13.23). PHBH obeys the following kinetic mechanism ... 

Figure 17. (A) The preparation of an electrically wired enzyme by the reconstitution technique, involving the removal of the native FAD cofactor from the enzyme (e.g., GOx) and the incorporation of the artificial FAD-ferrocene dyad into the apo-enzyme. (B) Cyclic voltammograms of a system consisting of ferrocene-FAD-reconstituted GOx (1.75 mg mL ) at various concentrations of glucose (a) 0, (b) 1, (c) 3 and (d) 20.5 mM. Experiments were performed in 0.1 M phosphate buffer, pH 7.3, at 35 C, using a cystamine-modified Au electrode, potential scan rate 2 mV s , under argon. Inset calibration curve of the biocatalytic current (0.5 V vs. SCE) at different glucose concentrations.

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