Nitroalkane oxidase

Gadda G, RD Edmondson, DH Russell, PF Fitzpatrick (1997) Identification of the naturally occurring flavin of nitroalkane oxidase from Fusarium oxysporum as a 5-nitrobutyl-FAD and conversion of the enzyme to the active FAD-containing form. J Biol Chem 111 5563-5570. 

Kido T, K Hashizume, K Soda (1978a) Purification and properties of nitroalkane oxidase from Fusarium oxysporium. J Bacterial 133 53-58. 

Daubner SC, G Gadda, Mp Valley, PF Fitzpatrick (2002) Cloning of nitroalkane oxidase from Fusarium oxysporum identifies a new member of the acyl-CoA dehydrogenase superfamily. Proc Natl Acad Sci USA 99 2702-2707. 

Fitzpatrick PF, Orville AM, Nagpal A, Valley MP. Nitroalkane oxidase, a carbanion-forming flavoprotein homologous to acyl-CoA dehydrogenase. Arch. Biochem. Biophys. 2005 433 157 -165. 

In this chapter I first summarize the theoretical methods developed in my group for enzyme kinetics modeling, which include both electronic and nuclear quantum effects. Then the methods are illustrated through applications to three energy systems, namely, alanine racemase, nitroalkane oxidase and dihydrofolate reductase. 

Classical (green) and quantum mechanical potential of mean force for the proton (red) and deuteron (blue) transfer from nitroethane to acetate ion in water (dashed curves) and to Asp402 (solid curves) at the active site of nitroalkane oxidase. 

The latter method, called the PI-FEP/UM approach, allows accurate primary and secondary kinetic isotope effects to be computed for enzymatic reactions. These methods are illustrated by applications to three enzyme systems, namely, the proton abstraction and reprotonation process catalyzed by alanine race-mase, the enhanced nuclear quantum effects in nitroalkane oxidase catalysis, and the temperature (in)dependence of the wild-type and the M42W/G121V double mutant of dihydrofolate dehydrogenase. These examples show that incorporation of quantum mechanical effects is essential for enzyme kinetics simulations and that the methods discussed in this chapter offer a great opportunity to more accurately model the mechanism and free energies of enzymatic reactions. 

Glucose oxidase and D- and L-amino acid oxidase accept nitroal-kane anions as substrates (20). The mechanism for flavoenzyme-catalyzed oxidation of nitroalkane has been established by Porter and Bright (20,21) to involve anN(5)-adduct as an intermediate as shown in... 

Equation 26. Electron-deficient flavins will also oxidize nitroalkane anions in model reactions (12). The observation (11) that nitromethane anion and FloXEt yield a stable 4a-adduct is evidence that 4a-adducts are not on the reaction path for nitroalkane oxidation. That the blocking of the N(5)-position of flavin (i.e., FloxEt) prevents oxidation of nitromethane would, however, be in accord with the requirement for an N(5)-adduct (11). The nitroalkane reaction with flavoenzyme has been used to implicate N(5)-adducts as intermediates in the oxidation mechanism of amino acid oxidases. However, it must be understood that nitroalkane anions differ significantly from the carbanions generated from a normal substrate. The nitroalkane anion on loss of its pair of electrons would provide an impossibly unstable carbonium ion, whereas in the case of the amino acid anion an internal electron release obviates carbonium ion formation. 

For these reasons, progress has been obtained with model, rather than physiological, substrates. In particular, recent studies of the reaction of the amino acid oxidases with )8-halogenated-a-amino acids and of D-amino acid oxidase and glucose oxidase with nitroalkanes and their carbanions have begun to clarify the chemical mechanism of these reactions. The results and interpretations of these studies are discussed briefly below. 

Studies of nitroalkane oxidation by n-amino acid oxidase (55) and glucose oxidase 49, 56) have provided strong evidence both for intermediate substrate carbanions and for subsequent covalent adduct formation between these and the N position of the flavin nucleus. The rationale for using nitroalkanes can be seen in the following reaction stoichiometries for D-amino acid oxidase (55) ... 

To understand the carbanion mechanism in flavocytochrome 62 it is useful to first consider work carried out on related flavoenzymes. An investigation into o-amino acid oxidase by Walsh et al. 107), revealed that pyruvate was produced as a by-product of the oxidation of )8-chloroalanine to chloropyruvate. This observation was interpreted as evidence for a mechanism in which the initial step was C -H abstraction to form a carbanion intermediate. This intermediate would then be oxidized to form chloropyruvate or would undergo halogen elimination to form an enamine with subsequent ketonization to yield pyruvate. The analogous reaction of lactate oxidase with jS-chlorolactate gave similar results 108) and it was proposed that these flavoenzymes worked by a common mechanism. Further evidence consistent with these proposals was obtained by inactivation studies of flavin oxidases with acetylenic substrates, wherein the carbanion intermediate can lead to an allenic carbanion, which can then form a stable covalent adduct with the flavin group 109). Finally, it was noted that preformed nitroalkane carbanions, such as ethane nitronate, acted as substrates of D-amino acid oxidase 110). Thus three lines of experimental evidence were consistent with a carbanion mechanism in flavoenzymes such as D-amino acid oxidase. 

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