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. Author manuscript; available in PMC: 2018 Oct 15.
Published in final edited form as: Arch Biochem Biophys. 2017 May 18;632:41–46. doi: 10.1016/j.abb.2017.05.012

Nitroalkane Oxidase: Structure and Mechanism

Paul F Fitzpatrick 1
PMCID: PMC5650508  NIHMSID: NIHMS882012  PMID: 28529198

Abstract

The flavoprotein nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes or ketones, releasing nitrite and transferring electrons to O2 to form H2O2. A combination of solution and structural analyses have provided a detailed understanding of the mechanism of this enzyme.

Keywords: Nitroalkane oxidase, flavoprotein, mechanism, structure, nitronate monooxygenase

Graphical Abstract

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The ability of flavoproteins to catalyze the oxidation of nitroalkanes was first described by Bright and coworkers as a side reaction of D- and L-amino acid oxidase and of glucose oxidase [1]. Subsequent mechanistic studies were consistent with these reactions involving the nitroalkane anion (nitronate), but with different mechanisms for D-amino acid oxidase (DAAO) and glucose oxidase [2, 3]. The mechanism proposed for oxidation of nitroethane anion by DAAO (Scheme 1) involved a nucleophilic attack of the substrate on N5 of the flavin, followed by loss of nitrite to form an electrophilic intermediate that was attacked by hydroxide. Collapse of the resulting tetrahedral intermediate would yield reduced flavin and acetaldehyde. This mechanism was supported by the ability to trap the initial tetrahedral adduct formed upon nitrite elimination with cyanide [2]and by kinetic isotope effects [4]. Studies of the oxidation of nitroethane by glucose oxidase established that the mechanism differed from that seen with DAAO, but the reaction was more complicated, with non-stoichiometric amounts of products. While the results were suggestive of the involvement of radicals, no definitive mechanism was proposed.

Scheme 1.

Scheme 1

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Oxidation of nitroalkane anions by D-amino acid oxidase.

Soon after the initial observation of nitroalkane oxidation by DAAO and glucose oxidase, the group of Soda showed that multiple strains of bacteria, yeasts, and fungi could grow on nitroethane as a nitrogen source, with nitrite accumulating in the medium [5]. They subsequently isolated flavoproteins from Hansenula mrakii and Fusarium oxysporum that were able to catalyze the oxidative denitrification of nitroalkanes [6, 7]. While both enzymes required oxygen, the enzyme from H. mrakii did not produce detectable amounts of hydrogen peroxide and produced superoxide as a transient intermediate [8]. In addition, this enzyme was more active on nitroalkane anions than neutral nitroalkanes [8]. Consequently, the enzyme, which was most active with 2-nitropropane, was labeled a 2-nitropropane dioxygenase [9]. In contrast, the enzyme from F. oxysporum produced a stoichiometric amount of hydrogen peroxide and nitrite, and was therefore labeled a nitroalkane oxidase (NAO) [7].

The initial characterizations of 2-nitropropane dioxygenase and NAO were consistent with different mechanisms that resembled the separate reactions of glucose oxidase and DAAO with nitroalkanes. The enzymes have different structures (Figure 1) and utilize different versions of the flavin cofactor, FMN for 2-nitropropane dioxygenase and FAD for NAO. Subsequent studies of the former by the Gadda group supported the mechanism shown in Scheme 2, with the anionic nitroalkane as the substrate and superoxide an intermediate [10, 11]. This enzyme, renamed nitronate monooxygenase to reflect these findings, has been reviewed recently and will not be discussed further here [10]. Recent advances in understanding the mechanism of NAO and the role of the protein structure in catalysis will be the subject of the present review.

Figure 1. Structures of (A) nitroalkane oxidase from F. oxysporum and (B) nitronate monooxygenase from Pseudomonas aeruginosa.

Figure 1

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Based on pdb files 2C12[12] and 4Q4K[13].

Scheme 2. Mechanism of nitronate monooxygenase.

Scheme 2

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Adapted from reference [11].

Mechanistic studies

NAO was first identified in F. oxysporum based on a large increase in the protein level when the cells were grown in the presence of nitroethane [7]. However, the purified enzyme did not show a typical flavin spectrum [7]. Subsequent isolation and characterization of the flavin established that the cofactor was in the form of an N5-nitrobutyl flavin adduct that could slowly decay to form oxidized FAD [14, 15]. The mechanism of Scheme 3 was proposed to explain the formation of the nitrobutyl-FAD based on the precedent with DAAO (Scheme 1). Consistent with such a mechanism, the addition of a mixture of neutral and anionic nitroethane to the FAD-containing enzyme regenerated the modified flavin.

Scheme 3.

Scheme 3

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Mechanism of formation of nitrobutyl flavin during turnover of nitroalkane oxidase with nitroethane.

Cloning of NAO from F. oxysporum identified it as homologous to the acyl-CoA dehydrogenase (ACAD) family of flavoproteins, with a sequence identity of 23–27% to individual family members [16, 17]. The mechanism of ACADs is proposed to involve abstraction of a proton from the α-carbon of the substrate as a hydride is transferred from the β-carbon to the flavin [18]. The active-site base in the ACAD family, a glutamate, aligns with Asp402 of NAO, implicating the latter as the active site base in that enzyme. The effects of mutating Asp402 (Table 1) [19] provided more direct evidence for Asp402 as the active-site base. Critically, the effect of mutating this residue could be bypassed by using nitroethane anion as the substrate [20].

Table 1.

Kinetic Parameters for Active Site Variants of F. oxysporum NAO with Nitroethane*

Enzyme kcat, s−1 kred, s−1 Km, mM kcat/Km, mM−1s−1
Wild-typea 15 ± 1 247 ± 5 2.3 ± 0.2 6.3 ± 0.4
S171Ab 5.4 ± 0.4 49 ± 12 2.1 ± 0.4 2.6 ± 0.3
S276Ac 1.0 ± 0.01 - 3.3 ± 0.2 0.30 ± 0.02
D402Ea 3.1 ± 0.1 16.3 ± 0.2 14.6 ± 0.8 0.21 ± 0.01
C397Sb 5.4 ± 0.4 42 ± 10 2.1 ± 0.4 2.6 ± 0.3
Y398Fb 11 ± 1 96 ± 33 4.8 ± 0.7 2.2 ± 0.2
R409Kd 2.6 ± 0.3 2.6 ± 0.3 26.3 ± 4.6 0.098 ± 0.006
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*

Conditions: pH 8.0, 30 °C.

a

From reference [19].

b

From reference [21].

c

From reference [22].

d

From reference [23]

In the mechanisms of Schemes 1 and 3, the nitroalkane anion, formed either in solution or in the active site, attacks N5 of the flavin. This is followed by nitrite elimination to form an electrophilic species that is attacked by a nucleophile, hydroxide in Scheme 1 and nitroethane anion in Scheme 3. When NAO was allowed to turn over with neutral nitroethane or 1-nitrohexane as substrate in the presence of cyanide, the enzyme lost activity and the flavin spectrum was altered to one consistent with an N5 adduct [24]. The mass spectrum of the isolated cofactor with both substrates supported its identification as the product of cyanide trapping the proposed cationic imine intermediate. The kinetic competency of this intermediate was established using deuterium kinetic isotope effects and rapid-reaction kinetics, eliminating the possibility that this species forms in a side reaction. Taken together, these results support the mechanism of Scheme 4 for NAO.

Scheme 4.

Scheme 4

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Mechanism of nitroalkane oxidase.

The mechanism in Scheme 4 is unique among flavoprotein oxidases in the involvement of the substrate anion and the subsequent nucleophilic attack on the oxidized flavin. A similar mechanism involving a carbanion intermediate was considered possible for flavoproteins oxidizing amines and alcohols [25, 26], but has been ruled out by more recent mechanistic and structural studies [27, 28].

The nonenzymatic version of the initial step in the NAO reaction, formation of a nitroalkane anion by proton abstraction, has been heavily studied as a model for understanding proton abstraction from carbon [29] and the contribution of quantum-mechanical tunneling to hydrogen transfer reactions [30]. Kinetic isotope effects established that proton abstraction from nitroethane is fully rate limiting for the reductive half-reaction of NAO [31], providing an opportunity to compare enzymatic and nonenzymatic proton abstraction from nitroethane. The temperature dependence of the kinetics of proton abstraction, including the deuterium kinetic isotope effects, by acetate, phosphate, and NAO were consistent with a significant decrease in ΔH‡ for the enzyme-catalyzed reaction, but did not provide evidence for a large contribution of tunneling [32]. A subsequent computational analysis using the path-integral free-energy approach suggested that the transmission coefficient for proton transfer is about three-fold greater in the enzymatic reaction [33, 34]. Solvent isotope effects have also been used to examine the mechanisms of the enzymatic and non-enzymatic reactions. Both the kcat/Km value for NAO with nitroethane as substrate [35] and the rate constant for nonenzymatic formation of nitroethane anion show solvent isotope effects of ~0.7 [36, 37]. This was ascribed to an effect of the solvent on the structure of nitroethane in solution rather than on the transition state for proton abstraction [35].

Structural studies

The first high-resolution structures of NAO to become available were of the resting oxidized enzyme and the enzyme containing the N5-nitrobutyl adduct [12]. The overall structure (Figure 1A) confirmed the homology with ACAD (Figure 2A), with RMSDs of less than 2 Å versus members of the ACAD family, and showed that Asp402 occupied the same location in the active site of NAO as Glu376 in medium chain ACAD (Figure 2B). Some of the subunits of active NAO contained a molecule of spermine in the active site from the crystallization buffer. The spermine had a similar position relative to the FAD and the active site base as the alkyl chain of the substrate of ACAD (Figure 2B), demonstrating that the substrates of the two enzymes bind in similar locations.

Figure 2. Comparison of the structures of MCAD and NAO.

Figure 2

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A) Structure of pig medium chain acylCoA dehydrogenase (PDB file 3MDE[38]). B) Overlays of the active sites of F. oxysporum nitroalkane oxidase D402N with spermine (tan carbons, PDB file 2C12[12]) and pig medium chain acylCoA dehydrogenase with 3-thiaoctanoyl-CoA (light blue carbons, PDB file 1UDY[39]).

The structure of NAO identified several active site residues that could play roles in the catalytic mechanism. In the active site of NAO without a ligand bound (Figure 3), one carboxylate oxygen of the active-site base Asp402 is at the appropriate distance from the hydroxyl of Ser276 to form a hydrogen bond, while the other carboxylate oxygen is positioned between the hydroxyl of Ser276 and the guanidino group of Arg409. The backbone carbonyl of Asp 402 forms a hydrogen bond with the same nitrogen of Arg409. The hydroxyl of Ser171 interacts with the flavin N5; in the ACAD family a conserved threonine shows a similar interaction. In addition, chemical modification studies had previously identified Cys397 [40] and Tyr398 [41], located near the xylene ring of the flavin, as possible active site residues. The contribution of each of these residues to catalysis has been examined by site-directed mutagenesis (Table 1). Mutagenesis of Asp402, Arg409, or Ser276 decreases the kcat/Km value for nitroethane and the rate constant for flavin reduction, kred, by about two orders of magnitude, consistent with their playing key roles in nitroalkane oxidation. In contrast the changes in the kinetic parameters upon mutagenesis of Ser171, Cys 297, or Tyr398 are 5-fold or less.

Figure 3.

Figure 3

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Active site residues in nitroalkane oxidase (PDB file 2C12[12]).

The low activity of the D402 variants made it possible to obtain structures of nitroalkanes bound to oxidized NAO. Figure 4 shows the active sites of D402N NAO with nitroethane, 1-nitrohexane, or 1-nitrooctane bound. The alkyl chains of nitrohexane and nitrooctane bind similarly to the spermine molecule seen in some subunits of the initial NAO structure (Figure 2). In all cases these extend up a hydrophobic tunnel from the active site to the surface. Analyses of the substrate specificity of NAO have established that the enzyme prefers linear primary nitroalkanes with longer alkyl chains [42], with the kcat/Km value increasing by 2.6 kcal/mol for each additional methylene group until a limiting value is reached with 1-nitrobutane. The nitro group of nitroethane is displaced from the nitro groups of the other two substrates in Figure 4. The combined kinetic and structural data suggest that alkyl chains must be long enough to extend into the active site tunnel for proper positioning of the substrate for catalysis. Consistent with that, isotope effects have shown that the 2.6 kcal/mol added by each methylene group contributes both to binding and to catalysis [43].

Figure 4. Overlay of structures of D402N nitroalkane oxidase with nitroethane (NE), 1-nitrohexane (NH) or 1-nitrooctane (NO) bound.

Figure 4

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From PDB files 3FCJ, 3D9D, and 3D9E [22, 33].

A key intermediate in the mechanism of Scheme 4 is the electrophilic imine intermediate. Critical evidence for the presence of this intermediate was the ability to trap this intermediate during turnover with 1-nitrohexane. Figure 5 shows the structure of the active site of the trapped enzyme, clearly showing the cyanohexyl flavin. The flavin is also clearly bent. The relative orientation of the active site residues is slightly altered from that seen in the substrate complexes, with Asp402 forming a hydrogen bond with Ser276 but not Arg409. The backbone amide of Asp402 forms hydrogen bonds with the nitrogen of the cyano group and the ribityl 2′ hydroxyl of the FAD. Finally, the side chain carbonyl of Asp402 forms a bifurcated hydrogen bond with two nitrogens of the guanidino group of Arg409. The structure of the protein containing the adduct also identified a possible path for loss of nitrite and entry of water into the active site (Figure 5), a channel containing a chain of hydrogen bonds from the flavin 2′ hydroxyl to the surface made up of water molecules and a glycerol molecule from solution.

Figure 5. Structure of nitroalkane oxidase trapped with cyanide during turnover with 1-nitrohexane.

Figure 5

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left, structure of active site containing cyanohexyl (green) flavin adduct; right, hydrogen bond network from active site to surface. Based on PDB file 3D9G [22].

Identification of new nitroalkane oxidases and discrimination from nitronate monooxygenases

The homology of NAO to ACAD makes it likely that a number of NAOs have inaccurately been annotated as ACADs. One significant difference between the two enzymes families is that NAO utilizes an aspartate residue as the active site base, whereas ACADs utilize a glutamate. This suggests that the two enzyme can readily be distinguished on this basis alone. To identify additional NAOs a PSI-Blast [44] search of sequence databases was carried out using the sequence of F. oxysporum NAO. Eliminating any sequences in which the residue aligning with Asp402 was a glutamate allowed identification of putative NAOs [45]. All of the candidates were fungal proteins. In addition all contained an arginine residue that aligned with Arg409 and a serine or threonine residue that aligned with Ser376 (Figure 6). The enzyme from Podospora anserina was expressed in E. coli and characterized. It catalyzed the oxidation of neutral nitroalkanes with reasonable kinetics (kcat/Km values of 104–105 M−1s−1). Mutagenesis of the aspartate, arginine, and serine corresponding to active site residues in F. oxysporum NAO decreased the activity significantly. Finally, the three-dimensional structure was determined, confirming it as an NAO. These results make it likely that other NAOs annotated as ACADs in the data base can be distinguished by a similar screen for aspartate as the active-site base.

Figure 6. Alignment of active-site residues in putative nitroalkane oxidases.

Figure 6

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Alignment of active site residues of F. oxysporum NAO with likely orthologs: NAO, F. oxysporum nitroalkane oxidase; PODANSg2158, Podospora anserina DSM 980 hypothetical protein; BC1G 11641, Botryotinia fuckeliana B05.10 hypothetical protein; SS1G 09730, Sclerotinia sclerotiorum 1980 hypothetical protein; NFIA 030710, Neosartorya fischeri putative acyl-CoA dehydrogenase; AN9162.2, Aspergillus nidulans hypothetical protein. Conserved residues are in bold and the active site catalytic triad Ser276, Asp402, and Arg409 in NAO are in bold and underlined. The numbering is for F. oxysporum NAO.

Two additional enzymes have recently been described as NAOs based on an ability to catalyze the oxidation of neutral nitroalkanes [46–48]. However, both the structural and kinetic properties of these enzymes are more consistent with their being nitronate monooxygenases rather than NAOs. The three-dimensional structures of both proteins show the same TIM-barrel fold as nitronate monooxygenases (Figure 1) [10, 13], and the cofactor in both is FMN rather than FAD. In the case of the enzyme from Streptomyces ansochromogenes, the reported kcat/Km value with neutral nitroethane is only 0.11 mM−1s−1 at 37 °C [46], far less than the value for F. oxysporum NAO at 30 °C (Table 1). In addition, some nitronate monooxygenases can utilize neutral nitroalkanes as slow substrates, but are far more active on the anions [10].

Conclusion

A comprehensive combination of mechanistic and structural approaches have provided a detailed understanding of the catalytic mechanism of NAO. Multiple structures are available with substrates bound, providing details of substrate interactions. The structure of NAO trapped with cyanide during turnover provides a structure of a key intermediate in turnover. Unlike the nitronate monooxygenases, NAO utilizes neutral nitroalkanes as substrates.

  • NAO catalyzes the oxidation of neutral nitroalkanes to the respective aldehyde or ketone and nitrite.

  • NAO has the same fold as acyl-CoA dehydrogenase.

  • NAO is unique in catalyzing the attack of the substrate anion on the flavin.

  • Protein structures are available of intermediates in catalysis, supporting the proposed mechanism.

Acknowledgments

The work from the author’s laboratory described here was supported in part by NIH grant GM058698.

Abbreviations used

NAO

nitroalkane oxidase

DAAO

D-amino acid oxidase

Footnotes

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