OYE homologues were found to catalyze quite different reactions: reductive denitrification of nitro-esters and nitro-aromatics, reduction of the aromatic ring of nitro-aromatics and the reduction of unsaturated !/"-bond in aldehydes and ketones. Some examples are shown in Figure 1.6. In many cases the physiological substrate is still unknown and not all enzymes are capable of catalyzing all reactions, but 2-cyclohexenone is observed to be a substrate in most OYE homologues. Although the substrates are very different, the overall reaction mechanism of OYE family members is similar. With only one binding site containing the non-covalently bound FMN, the enzymes act through a ping-pong Bi-Bi mechanism in which the cofactor NAD(P)H and the substrate use the same binding site. So the overall reaction proceeds via two steps - a reductive and an oxidative half-reaction, which can be individually analyzed by
rapid reaction techniques. All OYE homologues have characteristic UV-Vis spectra due to the conjugated double bonds of the isoalloxazine ring system of the cofactor. The spectra show peak maxima around 360 and 450 nm, depending on the protein environment of the FMN.
During reduction these maxima vanish and the protein becomes colorless. These spectral changes form the basis for rapid reaction measurements, such as stopped-flow spectrophotometry.
Figure 1.6: Different reactions catalyzed by OYE family members. (a) and (b): reductive denitrification of pentaerythritol tetranitrate (PETN) and glycerol trinitrate (nitroglycerine, GTN); (c): reduction of the aromatic ring of trinitrotoluene (TNT);
(d): reduction of 2-cyclohexenone; (e): reduction of morphinone (R = H) and codeinone (R = CH3); (f): reduction of N-ethylmaleimid (adapted from (Williams et al., 2004)).
Reductive and oxidative half-reactions have been extensively investigated for OYE1 (Massey
& Schopfer, 1986), morphinone reductase (Craig et al., 1998), PETN reductase (Khan et al., 2002) and YqjM (Fitzpatrick et al., 2004). In the first step - the reductive half-reaction - the enzyme is reduced by either NADH or NADPH. The physiological reductant of many OYE family members is assumed to be NADPH, whereas NemA and morphinone reductase prefer NADH. The mechanism by which the enzymes discern between the two nicotinamides is yet unknown (Brige et al., 2006). With stopped-flow measurements it is possible to distinguish between kinetically individual steps during reduction (see Figure 1.7a). For OYE1 two
oxidized enzyme intermediates were observed before the reduction of enzyme-bound flavin takes place. The first step is the binding of NAD(P)H and the formation of a Michaelis-complex. This initial binding step is followed by a charge-transfer complex between the FMN (charge-transfer acceptor) and NAD(P)H (charge-transfer donor). Finally, the reduced enzyme is formed by the reduction of the flavin and the release of NAD(P)+. In the case of PETN reductase, morphinone reductase and YqjM the discrete binding step prior to the charge-transfer complex formation was not observed.
Figure 1.7: General kinetic scheme for (a) reductive and (b) oxidative half-reactions of OYE family members. The initial formation of a Michaelis-complex was only observed for OYE1 but not for PETN reductase, morphinone reductase and YqjM.
The oxidative half-reactions of OYE homologues comprise the reduction of different types of substrates. Each enzyme is capable of reducing many different substrates more or less efficiently. 2-cyclohexenone was used as model substrate for the investigation of the oxidative half-reaction of OYE1 (Kohli & Massey, 1998), PETN reductase (Khan et al., 2002) and YqjM (Fitzpatrick et al., 2004). For morphinone reductase the reaction was first performed with the physiological substrate codeinone (Craig et al., 1998) and later with 2-cyclohexenone (Messiha et al., 2005). As in the reductive half-reaction, several individual steps could be discerned for the homologues in the oxidative half-reaction (see Figure 1.7b).
In morphinone reductase three resolvable steps were observed during reduction of codeinone.
The first was the formation of a charge-transfer complex between reduced FMN and substrate, followed by flavin reoxidation and finally the release of hydrocodone from the oxidized enzyme. However, for the reaction of 2-cyclohexenone with reduced PETN and YqjM no initial charge-transfer formation or product release were observed. Thus, a one step reaction mechanism was assumed to evaluate the kinetic data.
The reaction mechanisms of oxidative half-reactions of OYE homologues have been studied in detail for different substrates. In Figure 1.8 a general scheme for the reaction mechanism of the oxidative half-reaction with 2-cyclohexenone is shown.
For example the reduction of different !/"-unsaturated carbonyls by OYE1 was first investigated by Vaz and co-workers in 1995 (Vaz et al., 1995). Different aldehydes, ketones,
esters, amides, nitriles and acids were tested for their ability to oxidize the reduced enzyme.
However, only aldehydes and ketones acted as oxidative substrates. The carbonyl groups of these compounds are more basic than those of esters and amides and than cyano groups.
Therefore, hydrogen-bonding interactions between the carbonyl oxygen and an active site residue, like His191 in OYE1, increase the electrophilicity of the "-carbon towards the flavin hydride. To prove the hydride transfer from the N(5) of FMN, Vaz and co-workers tested different alkyl substituents at the !- and "-carbon of the substrates. They found that only substituents in "-position decrease the rate for hydride transfer to the olefinic bond, because of the steric hindrance. Hence the hydride transfer occurs from the reduced FMN to the "-carbon of the substrate (Vaz et al., 1995).
The putative proton donor to the !-carbon in OYE1 could be identified using site directed mutagenesis of active site residues as Tyr196 (Kohli & Massey, 1998). In morphinone reductase, the identification of the possible proton donor failed and Messiha and co-workers infer the solvent as proton source (Messiha et al., 2005). The same assumption was drawn for PETN reductase, where the corresponding residue Tyr186 was also shown not to be involved in a rate-limiting step (Khan et al., 2005).
Figure 1.8: Proposed reaction scheme of the oxidative half-reaction of OYE family members.
In the first step the !/"-unsaturated double bond is polarized by binding to active site residues. This enables the hydride transfer from flavin, followed by the proton addition from an active site residue or the solvent. (adapted from (Messiha et al., 2005))