Crystal Structure of 2-Nitropropane Dioxygenase Complexed with FMN and Substrate

Abstract

Nitroalkane compounds are widely used in chemical industry and are also produced by microorganisms and plants. Some nitroalkanes have been demonstrated to be carcinogenic, and enzymatic oxidation of nitroalkanes is of considerable interest. 2-Nitropropane dioxygenases from Neurospora crassa and Williopsis mrakii (Hansenula mrakii), members of one family of the nitroalkane-oxidizing enzymes, contain FMN and FAD, respectively. The enzymatic oxidation of nitroalkanes by 2-nitropropane dioxygenase operates by an oxidase-style catalytic mechanism, which was recently shown to involve the formation of an anionic flavin semiquinone. This represents a unique case in which an anionic flavin semiquinone has been experimentally observed in the catalytic pathway for oxidation catalyzed by a flavin-dependent enzyme. Here we report the first crystal structure of 2-nitropropane dioxygenase from Pseudomonas aeruginosa in two forms: a binary complex with FMN and a ternary complex with both FMN and 2-nitropropane. The structure identifies His 152 as the proposed catalytic base, thus providing a structural framework for a better understanding of the catalytic mechanism.Nitroalkanes are widely used in industry, because they are useful as intermediate compounds in chemical synthesis (1, 2). They are also synthesized by various organisms. Many antibiotics, e.g. chloramphenicol and azomycin, contain nitro groups, and many leguminous plants produce nitro toxins such as 3-nitro-1-propionic acid and 3-nitro-1-propanol (3). However, many nitroalkanes are expected to be toxic, and some have been shown to be carcinogenic (4 -10). For example, 2-nitropropane causes the formation of both 8-hydroxy-and 8-aminoguanine in the DNA and RNA (11). The enzymatic oxidation of nitroalkanes into less toxic species can therefore be exploited for use in bioremediation.2-Nitropropane dioxygenase (EC 1.13.11.32), one of the nitroalkaneoxidizing enzyme families, catalyzes oxidative denitrification of nitroalkanes to their corresponding carbonyl compounds and nitrites. To date, 2-nitropropane dioxygenase has been isolated from a fungus Neurospora crassa (12) and a yeast Williopsis mrakii (Hansenula mrakii) (13).The two enzymes have similar molecular masses of ϳ40 kDa, but their prosthetic groups are different. FMN and FAD are found in the N. crassa and W. mrakii (H. mrakii) enzymes, respectively (14, 15). The ncd-2 gene encoding for 2-nitropropane dioxygenase in N. crassa has been cloned and expressed in Escherichia coli (16). The heterologously expressed enzyme was found to be a homodimer containing 1 mol of non-covalently bound FMN per mole of subunit (16). A steady-state kinetic analysis showed that the preferred substrates for the enzyme are anionic nitronates as compared with neutral nitroalkanes and that the enzyme has broad substrate specificity that is independent of substrate size (16).It has been shown that 2-nitropropane dioxygenase operates through an oxidase-style catalytic mechanism, in which substrate oxidation occurs prior to and independently ...

Functional Annotation of a Presumed Nitronate Monoxygenase Reveals a New Class of NADH:Quinone Reductases

“…This study aimed to characterize PA1024, one of the three genes encoding a hypothetical NMO in P. aeruginosa PAO1. The current classification of PA1024 is based on a 2006 study (7), but the enzyme does not carry the consensus motifs characteristic of the recently defined class I and class II NMOs (6). The biochemical and kinetic characterization of PA1024 presented herein shows that the enzyme indeed is not an NMO, as indicated by the lack of oxygen consumption with propionate-3-nitronate and other nitronates.…”

“…To investigate this hypothesis, we experimentally tested NADH, NADPH, and various quinones for catalytic activity. Furthermore, we reanalyzed and modified the conserved motifs identified by Ha et al (7) in the protein sequence of PA1024, and we showed that they are present in more than 490 sequences in the non-redundant protein database. The results reinforce the need for accurate experimental data on select hypothetical proteins to work in concert with computational methods for improved gene function prediction.…”

“…P. aeruginosa PAO1 possesses two other genes coding for hypothetical NMOs, namely pa0660 and pa1024; however, these proteins do not carry the four motifs characteristic of class I NMO nor any of the motif signatures of class II NMO, raising the possibility that they code for enzymes with different function. Although there is no experimental evidence at transcript or protein levels for the gene product PA0660, a crystal structure for the hypothetical protein PA1024 is available for the enzyme in free form and in complex with 2-nitropropane (PDB codes 2GJL and 2GJN) at 2.0 and 2.3 Å resolution, respectively (7). The protein PA1024 is currently classified as 2-nitropropane dioxygenase, a previous name for NMOs, based on the gene function prediction (ϳ32% sequence identity) and a qualitative enzymatic assay performed with 20 mM 2-nitropropane at pH 6.5 (7).…”

“…Although there is no experimental evidence at transcript or protein levels for the gene product PA0660, a crystal structure for the hypothetical protein PA1024 is available for the enzyme in free form and in complex with 2-nitropropane (PDB codes 2GJL and 2GJN) at 2.0 and 2.3 Å resolution, respectively (7). The protein PA1024 is currently classified as 2-nitropropane dioxygenase, a previous name for NMOs, based on the gene function prediction (ϳ32% sequence identity) and a qualitative enzymatic assay performed with 20 mM 2-nitropropane at pH 6.5 (7). Nonetheless, the kinetic parameters with 2-nitropropane were not determined, and the physiological substrate P3N was not tested, because it was unknown at the time of the study.…”

Molecular Modeling and Docking Study of 2-Nitropropane Dioxygenase of Mycobacterium tuberculosis

“…It is essential for fatty acid synthesis and hence a potential target for the drugs against tuberculosis. The structure of Mycobacterium tuberculosis 2NPD enzyme is unknown [5,6].…”