A variety of enzymes that transform 3NPA and P3N (Table ) have been purified from eukaryotes. All are flavoproteins with either FMN or FAD as a tightly bound flavin cofactor. The subunit sizes fall within a narrow range, but the number of subunits of the functional enzyme varies. Nitronate monooxygenase (NMO; EC 126.96.36.199, formerly called 2-nitropropane dioxygenase, EC 188.8.131.52) and nitroalkane oxidase (NAO; EC 184.108.40.206) have broad substrate ranges but greatly prefer nitroalkanes to nitropropionic acid (15
). P3NO and NPAO have much narrower substrate ranges and are much more active with nitropropionic acid than with nitroalkanes (19
). All the enzymes that preferentially attack 3NPA release twice as much nitrate as nitrite from 3NPA, whereas reactions catalyzed by NMO and NAO release no nitrate. The striking similarity of the substrates and products of the PnoA-catalyzed reaction to those of the P3NO-catalyzed reaction from Penicillium atrovenetum
) suggests that PnoA is a P3NO. However, the lack of stoichiometric release of H2
indicates that the enzyme is a monooxygenase (13
), and we have tentatively designated it P3N monooxygenase. Full characterization of P3N monooxygenase is currently under way to rigorously establish the reaction mechanism and thus the proper EC designation.
Several lines of evidence indicate that the P3N monooxygenase does not catalyze the first step in the pathway. The fact that acetate-grown cells transform P3N but not 3NPA whereas 3NPA-grown cells transform both forms suggests that the P3N monooxygenase is constitutive. Neither cell extracts nor purified PnoA transformed 3NPA. The results suggest that the initial step in 3NPA degradation is transformation of 3NPA to the nitronate by an unidentified enzyme that is upregulated by growth on 3NPA. It is possible that the failure of acetate-grown cells to transform 3NPA indicates a transport problem rather than lack of induction of an enzyme. Even if that were so, the specificity of cell extracts and purified PnoA for P3N still would suggest a requirement for an enzyme in the 3NPA degradation pathway to catalyze the initial conversion to the nitronate. NMO (Table ) from Neurospora crassa
) is the only enzyme that readily transforms both nitronate and neutral forms of nitroalkanes, although nitronates are the preferred substrates. When the neutral nitroalkane is the substrate, the initial step after enzyme-substrate complex formation is removal of a proton to convert the substrate to a nitronate (11
). Thus, N. crassa
NMO has the function of both tautomerase and dioxygenase.
The lack of ammonia accumulation during growth of bacteria on 3NPA indicates that 3NPA is not reduced to β-alanine as in cattle and sheep rumen (4
). Purified PnoA catalyzed transformation of P3N with the release of nitrate and nitrite in a 2:1 ratio, whereas growing cells released only 2% of the nitrogen as nitrite. The observations suggest that JS189 and JS190 incorporate nitrite as the nitrogen source, which accounts for the missing nitrogen in culture fluids.
MSA is an important intermediate in multiple anabolic and catabolic pathways, and a number of enzymes that transform MSA have been described (Fig. ). The lack of MSA decarboxylase activity was due to a lack of expression of msaD
, which is reminiscent of the myo
-inositol degradation pathway in Lactobacillus casei
). The myo
-inositol operon carries genes for MSA oxidative decarboxylase (iolA
) and MSA decarboxylase (iolK
), but only iolA
Based on the above results, we propose that the pathway for degradation of 3NPA (Fig. ) is initiated by an inducible, but as yet unidentified, enzyme that converts 3NPA to P3N. The key step in the pathway is the denitration of P3N by the action of a constitutive P3N monooxygenase, encoded by the pnoA gene. An inducible MSA oxidative decarboxylase then converts MSA to acetyl-CoA, which enters central metabolic pathways. Additional sequencing will be required to locate the genes that encode the MSA oxidative decarboxylase and the hypothesized initial enzyme. The facile isolation of bacteria that grow on 3NPA suggests a highly evolved and widespread degradation pathway that may have evolved to exploit plant or fungal production of 3NPA.
The function of all other enzymes that transform 3NPA and nitroalkanes has been ascribed to detoxification or protection, but their physiological roles have not been established. P3NO and 3NPAO have been found only in organisms that also produce 3NPA. The presence of 3NPA in plants has been attributed to antiherbivory strategies, while the presence of 3NPA in fungi remains to be explained. In contrast to the other enzymes, PnoA in Cupriavidus sp. JS190 and Pseudomonas sp. JS189 clearly serves as a means to exploit 3NPA as a growth substrate. The P3N monooxygenase described here is the only member of the group whose physiological role has been established and the first P3N monooxygenase for which a gene sequence has been reported.
Gene sequences that encode dioxygenases related to 2-nitropropane dioxygenase constitute COG2070, which comprises 53 proteins distributed among 30 genomes (http://www.ncbi.nlm.nih.gov/COG/grace/wiew.cgi?COG2070
), with over 2,100 nucleotide sequences in GenBank (Fig. ). Many organisms contain multiple genes annotated as encoding 2-nitropropane dioxygenase. Despite the apparent widespread distribution of the COG, prior to this study, only proteins from Williopsis saturnus
(formerly Hansenula mrakii
), Neurospora crassa
), Pseudomonas aeruginosa
), and Streptomyces achromogenes
) had been purified and characterized to various degrees. Although called 2-nitropropane dioxygenase, no evidence established 2-nitropropane as the physiological substrate of any of the enzymes included in COG2070. The recent reclassification of 2-nitropropane dioxygenase as nitronate monooxygenase (13
) was based on the recharacterization of purified proteins from the ascomycetes.
FIG. 5. Distribution of selected members of COG2070 based on amino acid sequences of 2-nitropropane dioxygenases and P3N monooxygenase. Boldface lettering denotes biochemically characterized enzymes. Asterisks denote P3N monooxygenases identified in the present (more ...)
The P3N monooxygenase is biochemically distinct from the well-characterized nitronate monooxygenases and falls within COG2070, but with only ~20 to 25% amino acid identity to the biochemically characterized enzymes. Many of the current annotations are wrong (12
), and the situation with 2-nitropropane dioxygenase seems to be another example of annotation based only on modest sequence similarity without functional information. Our preliminary investigation of putative nitronate monooxygenases from B. phytofirmans
PsJN and P. aeruginosa
PAO1 confirms that the three closely related genes encode P3N monooxygenase rather than 2-nitropropane dioxygenase, based on the specificity of the enzymes for P3N and the lack of activity for 2-nitropropane. Ha et al. crystallized a 2-nitropropane dioxygenase from P. aeruginosa
PAO1 and analyzed and aligned the amino acid sequence with several closely related enzymes (16
) but did not determine the physiological substrate of the enzyme. Six motifs were described, and 10 highly conserved residues that interact with FMN were identified. When the P3N monooxygenases described here were added to the alignment, only three of the highly conserved residues interacting with FMN along with the His152
identified as the catalytic base were conserved across both 2-nitropropane dioxygenase and P3N monooxygenase (Fig. ). Motifs II and IV, the latter being described as the most highly conserved motif by Ha et al., are disrupted in the P3N monooxygenases. Taken together, the evidence suggests that P3N monooxygenases form a separate cluster within COG2070 and that many of the proteins annotated as “2-nitropropane dioxygenase” are in fact P3N monooxygenases (Fig. ). The argument is supported by the fact that 3NPA is much more likely to be widespread in natural ecosystems than are 2-nitropropane and other nitroalkanes.
FIG. 6. Alignment of 2-nitropropane dioxygenases and P3N monooxygenase. Sequences 1 to 7 were analyzed by Ha et al. (16). The motifs (colored blocks) are highly conserved residues that interact with FMN (red circles). An inverted green triangle represents the (more ...)
The facile isolation of bacteria that grow on 3NPA suggests a highly evolved and widespread degradation pathway that may have evolved to exploit plant or fungal production of 3NPA. The ability of the pnoA
-containing strains to grow on 3NPA as a nitrogen source but not always as a carbon source suggests the lack of a complete degradation pathway or an inability to regulate such a pathway in some strains. It is possible that, in some bacteria, the presence of P3N monooxygenase might be a detoxification mechanism similar to the proposed function in fungi and plants. It is also a plausible mechanism of scavenging nitrogen. There may be a continuum of enzymes with functions from protection to growth represented in the diversity of genes in COG2070. The 3NPA-degrading bacteria are the only organisms reported to grow well on 3NPA as a sole source of carbon, nitrogen, and energy. An Alcaligenes
sp. was reported to produce nitrate and nitrite from 3NPA; however, after 28 days, only a fraction of the initial 3NPA was consumed, and minimal growth was observed (7
). The failure of E. coli
to either grow on or be inhibited by 3NPA suggests alternate means of detoxification of 3NPA or insensitivity to its effects. We are currently more fully characterizing the biochemistry of the P3N monooxygenase and ecological roles of bacteria that degrade 3NPA.