Kevin Francis

Nitronate monooxygenase, a model for anionic flavin semiquinone intermediates in oxidative catalysis

Nitronate monooxygenase (NMO), formerly referred to as 2-nitropropane dioxygenase, is an FMN-dependent enzyme that uses molecular oxygen to oxidize (anionic) alkyl nitronates and, in the case of the enzyme from Neurospora crassa, (neutral) nitroalkanes to the corresponding carbonyl compounds and nitrite. Over the past 5 years, a resurgence of interest on the enzymology of NMO has driven several studies aimed at the elucidation of the mechanistic and structural properties of the enzyme. This review article summarizes the knowledge gained from these studies on NMO, which has been emerging as a model system for the investigation of anionic flavosemiquinone intermediates in the oxidative catalysis of organic molecules, and for the effect that branching of reaction intermediates has on both the kinetic parameters and isotope effects associated with enzymatic reactions. A comparison of the catalytic mechanism of NMO with other flavin-dependent enzymes that oxidize nitroalkane and nitronates is also presented.

The biochemistry of the metabolic poison propionate 3‐nitronate and its conjugate acid, 3‐nitropropionate

3‐Nitropropionate (3‐NPA) is a nitro aliphatic compound found in numerous plants and fungi. The nitro compound exists in equilibrium with its conjugate base, propionate 3‐nitronate (P3N) and has a pKa approaching the physiological range of 9.1. Since 1920, more than 30 species of plant and fungi have been identified as producing 3‐NPA as a means of defense from herbivores. Glycoside products containing moieties of 3‐NPA found in parts of the plants most accessible to herbivores can be easily hydrolyzed to free 3‐NPA by bacterial enzymes in the gut of animals. In addition to providing a defense mechanism, the nitro compound is an intermediate in the nitrification process of leguminous plants. The synthesis of 3‐NPA in these plants and fungi is poorly understood. P3N, which readily forms from 3‐NPA at physiological pH, is a potent inhibitor of the key enzyme succinate dehydrogenase in the Krebs cycle and electron transport chain. Inhibition of succinate dehydrogenase in humans and livestock causes neurotoxicity and in some cases death. Several enzymes catalyze the oxidation of 3‐NPA or P3N; all contain a noncovalently bound flavin cofactor and are found in the organisms that produce 3‐NPA. With kcat/Km values of >106 M−1 s−1, nitronate monooxygenases can quickly and efficiently oxidize P3N to malonic semialdehyde as a means of protecting the organism from killing itself. Although it was discovered almost a century ago, the biochemistry and physiological role of 3‐NPA/P3N are just emerging.

A novel activity for fungal nitronate monooxygenase: detoxification of the metabolic inhibitor propionate-3-nitronate.

Nitronate monooxygenase (NMO; E.C. 1.13.12.16) oxidizes alkyl nitronates to aldehydes and nitrite. Although the biochemistry of the enzyme from fungal sources has been studied extensively, the physiological role is unknown. The ability of NMO to detoxify propionate-3-nitronate was tested by measuring growth of recombinant Escherichia coli containing the gene encoding for the enzyme in either the absence or presence of the nitronate and its conjugate acid 3-nitropropionate. The mixture propionate-3-nitronate/3-nitropropionate is toxic to E. coli cells lacking expression of NMO, but the toxicity is overcome through either induction of the gene for NMO or through addition of exogenous enzyme to the cultures. Both Williopsis saturnus and Neurospora crassa were able to grow in the presence of 0.4mM propionate-3-nitronate and 19.6mM 3-nitropropionate, while a knockout mutant of N. crassa lacking NMO was inhibited by concentrations of propionate-3-nitronate and 3-nitropropionate >0.3 and 600μM, respectively. These results strongly support the conclusion that NMO functions to protect the fungi from the environmental occurrence of the metabolic toxin.

Inflated kinetic isotope effects in the branched mechanism of Neurospora crassa 2-nitropropane dioxygenase.

Catalytic turnover of Neurospora crassa 2-nitropropane dioxygenase with nitroethane as substrate occurs through both nonoxidative and oxidative pathways. The pH dependence of the kinetic isotope effects with [1,1-(2)H(2)]nitroethane as substrate was measured in the current study by monitoring the formation of the nitronate product in the nonoxidative pathway. The kinetic isotope effect on the second-order rate constant for nitronate formation, k(cat)/K(m), decreased from an upper limiting value of 23 +/- 1 at low pH to a lower limiting value of 11 +/- 1 at high pH. These kinetic isotope effects are three times larger than those determined previously through measurements of oxygen consumption that occurs in the oxidative pathway of the enzyme [(2006) Biochemistry 45, 13889]. Analytical expressions for the k(cat)/K(m) values determined in each study show that the difference in the kinetic isotope effects arises from the branching of an enzyme-ethylnitronate reaction intermediate through oxidative and nonoxidative turnover. This branching is isotope sensitive due to a kinetic isotope effect on nitronate release rather than on flavin reduction as indicated by the pH-independent (D)k(red) value of 0.99 +/- 0.06 with ethylnitronate as substrate. The kinetic isotope effect on ethylnitronate release arises from the deprotonation of histidine 196, which provides electrostatic interactions with the nitronate to keep it bound in the active site for oxidation. The isotope effect on branching results in an inflation of the kinetic isotope observed for the nonoxidative pathway to values that are larger than the intrinsic values associated with CH bond cleavage.

Kinetic evidence for an anion binding pocket in the active site of nitronate monooxygenase.

A series of monovalent, inorganic anions and aliphatic aldehydes were tested as inhibitors for Hansenula mrakii and Neurospora crassa nitronate monooxygenase, formerly known as 2-nitropropane dioxygenase, to investigate the structural features that contribute to the binding of the anionic nitronate substrates to the enzymes. A linear correlation between the volumes of the inorganic anions and their effectiveness as competitive inhibitors of the enzymes was observed in a plot of pK(is)versus the ionic volume of the anion with slopes of 0.041+/-0.001 mM/A(3) and 0.027+/-0.001 mM/A(3) for the H. mrakii and N. crassa enzymes, respectively. Aliphatic aldehydes were weak competitive inhibitors of the enzymes, with inhibition constants that are independent of their alkyl chain lengths. The reductive half reactions of H. mrakii nitronate monooxygenase with primary nitronates containing two to four carbon atoms all showed apparent K(d) values of approximately 5 mM. These results are consistent with the presence of an anion binding pocket in the active site of nitronate monooxygenase that interacts with the nitro group of the substrate, and suggest a minimal contribution of the hydrocarbon chain of the nitronates to the binding of the ligands to the enzyme.