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 His152 as the proposed catalytic base, thus providing a structural framework for a better understanding of the catalytic mechanism. 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 His152 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 (1Ballini R. Barboni L. Giarlo G. J. Org. Chem. 2003; 68: 9173-9176Crossref PubMed Scopus (31) Google Scholar, 2Hanessian C. Pham V. Org. Lett. 2000; 2: 2975-2978Crossref PubMed Scopus (322) Google Scholar). 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 (3Kido T. Tanizawa K. Inagaki K. Yoshimura T. Ishida M. Hashizume K. Soda K. Agric. Biol. Chem. 1984; 48: 2549-2554Google Scholar). However, many nitroalkanes are expected to be toxic, and some have been shown to be carcinogenic (4Davis R. Clayton G. Clayton F. Patty's Industrial Hygiene and Toxicology. II. 1993: 559-662Google Scholar, 5Sittig M. Handbook of Toxic and Hazardous Chemicals and Carcinogens, 3rd Ed. 1991; (pp. ; 1214-1215; 1225-1226, 1226-1228, Noyes Publications, Park Ridge, NJ): 1198-1199Google Scholar, 6Haas-Jobelius M. Coulston F. Korte F. Ecotoxicol. Environ. Saf. 1992; 23: 253-259Crossref PubMed Scopus (13) Google Scholar, 7Fiala E.S. Czerniak R. Castonguay A. Conaway C.C. Rivenson A. Carcinogenesis. 1987; 8: 1947-1949Crossref PubMed Scopus (75) Google Scholar, 8Lewis T.R. Ulrich C.E. Busey W.M. J. Environ. Pathol. Toxicol. 1979; 2: 233-249PubMed Google Scholar, 9Dayal R. Gescher A. Harpur E.S. Pratt I. Chipman J.K. Fundam. Appl. Toxicol. 1989; 13: 341-348Crossref PubMed Google Scholar, 10Conaway C.C. Nie G. Hussain N.S. Fiala E.S. Cancer Res. 1991; 51: 3143-3147PubMed Google Scholar). For example, 2-nitropropane causes the formation of both 8-hydroxy- and 8-aminoguanine in the DNA and RNA (11Sakano K. Oikawa S. Murata M. Hiraku Y. Kojima N. Kawanishi S. Mutat. Res. 2001; 479: 101-111Crossref PubMed Scopus (30) Google Scholar). 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 (12Little H.N. J. Biol. Chem. 1951; 193: 347-358Abstract Full Text PDF PubMed Google Scholar) and a yeast Williopsis mrakii (Hansenula mrakii) (13Tchorzewski M. Kurihara T. Esaki N. Soda K. Eur. J. Biochem. 1994; 226: 841-846Crossref PubMed Scopus (11) Google Scholar). The two enzymes have similar molecular masses of ∼40 kDa, but their prosthetic groups are different. FMN and FAD are found in the N. crassa and W. mrakii (H. mrakii) enzymes, respectively (14Gorlatova N. Tchorzewski M. Kurihara T. Soda K. Esaki N. Appl. Environ. Microbiol. 1998; 64: 1029-1033Crossref PubMed Google Scholar, 15Kido T. Soda K. J. Biol. Chem. 1978; 253: 226-232Abstract Full Text PDF PubMed Google Scholar). The ncd-2 gene encoding for 2-nitropropane dioxygenase in N. crassa has been cloned and expressed in Escherichia coli (16Francis K. Russell B. Gadda G. J. Biol. Chem. 2005; 280: 5195-5204Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The heterologously expressed enzyme was found to be a homodimer containing 1 mol of non-covalently bound FMN per mole of subunit (16Francis K. Russell B. Gadda G. J. Biol. Chem. 2005; 280: 5195-5204Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). 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 (16Francis K. Russell B. Gadda G. J. Biol. Chem. 2005; 280: 5195-5204Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). It has been shown that 2-nitropropane dioxygenase operates through an oxidase-style catalytic mechanism, in which substrate oxidation occurs prior to and independently from reaction with oxygen, via the formation of an enzyme-bound anionic flavin semiquinone (16Francis K. Russell B. Gadda G. J. Biol. Chem. 2005; 280: 5195-5204Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). This represents the first account in which an anionic flavin semiquinone has been experimentally observed in the catalytic pathway for the oxidation of organic molecules catalyzed by a flavin-dependent enzyme (16Francis K. Russell B. Gadda G. J. Biol. Chem. 2005; 280: 5195-5204Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Despite many interesting features, no three-dimensional structure has yet been reported for any 2-nitropropane dioxygenase. Because all previous biochemical works have been done on the enzymes from N. crassa and W. mrakii (H. mrakii), the genomic DNAs of N. crassa and W. mrakii (H. mrakii) were not commercially available. Therefore, we chose 2-nitropropane dioxygenase from Pseudomonas aeruginosa as part of the laboratory-scale structural genomics project. P. aeruginosa 2-nitropropane dioxygenase is a 328-residue protein (Mr 34,819), showing 23% sequence identity to 2-nitropropane dioxygenase from N. crassa. It also shows significant levels of sequence similarity to putative 2-nitropropane dioxygenases from various sources. In this study, we have overexpressed 2-nitropropane dioxygenase from P. aeruginosa in E. coli, crystallized it, and solved its structure, representing the first structure of any 2-nitropropane dioxygenase. We describe the structure in two forms: (i) a binary complex with FMN and (ii) a ternary complex with FMN and 2-nitropropane. This study provides detailed structural information on the binding mode of the cofactor and substrate. The structure also suggests that the highly conserved His152 is the likely candidate for the catalytic base, thus allowing a better understanding of the catalytic mechanism of 2-nitropropane dioxygenase family. Protein Expression and Purification—The PA1024 gene encoding 2-nitropropane dioxygenase from P. aeruginosa was amplified by PCR using the genomic DNA of P. aeruginosa strain PAO1 as template. The forward and reverse oligonucleotide primers were designed using the published genome sequence (17Stover C.K. Pham X.Q. Erwin A.L. Mizoguchi S.D. Warrener P. Hickey M.J. Brinkman F.S. Hufnagle W.O. Kowalik D.J. Lagrou M. Garber R.L. Goltry L. Tolentino E. Westbrock-Wadman S. Yuan Y. Brody L.L. Coulter S.N. Folger K.R. Kas A. Larbig K. Lim R. Smith K. Spencer D. Wong G.K. Wu Z. Paulsen I.T. Reizer J. Saier M.H. Hancock R.E. Lory S. Olson M.V. Nature. 2000; 406: 959-964Crossref PubMed Scopus (3325) Google Scholar) as 5′-G GAA TTC CAT ATG GGC GTG TTC CGT ACC CGT TT-3′ and 5′-CCG CCG CTC GAG GAC CCC GGC GAG CAT GCC C-3′, respectively. The underlined bases denote the NdeI and XhoI cleavage sites. The PCR product was digested with NdeI and XhoI and was then inserted into the NdeI/XhoI-digested expression vector pET-21a(+) (Novagen). This vector construction adds an eight-residue tag (LEHHHHHH) to the C terminus of the gene product to facilitate protein purification. The recombinant enzyme was expressed in E. coli C41(DE3) cells (18Miroux B. Walker J.E. J. Mol. Biol. 1996; 260: 289-298Crossref PubMed Scopus (1546) Google Scholar) using Luria-Bertani broth. Protein expression was induced by 1 mm isopropyl 1-thio-β-d-galactopyranoside, and the cells were incubated for an additional 22 h at 20 °C following growth to mid-log phase at 37 °C. The cells were lysed by sonication in a lysis buffer (20 mm Tris-HCl at pH 7.9, 500 mm NaCl) containing 50 mm imidazole. The crude lysate was centrifuged at ∼36,000 × g for 60 min. The supernatant was applied to a Ni+-chelated HiTrap chelating column (Amersham Biosciences). The protein was eluted with the lysis buffer containing 500 mm imidazole. Next, gel filtration was performed on a HiLoad XK 16 Superdex 200 prep-grade column (Amersham Biosciences) that was previously equilibrated with buffer A (50 mm Tris-HCl, pH 7.5) containing 100 mm sodium chloride. At the third chromatography step, the protein was loaded onto a mono Q HR5/5 ion-exchange column (Amersham Biosciences) that was previously equilibrated with buffer A, and the protein was eluted with a linear gradient of 0-1.0 m sodium chloride. The selenomethionine (SeMet) 3The abbreviations used are: SeMet, selenomethionine; MAD, multiwavelength anomalous diffraction; PDB, Protein Data Bank; r.m.s., root mean square. -substituted protein was expressed in E. coli B834(DE3) cells, using the M9 cell culture medium containing extra amino acids. The procedure for purifying the SeMet-substituted protein was the same except for the presence of 10 mm dithiothreitol in all buffers used during purification steps. Enzyme Assay and Mutagenesis—2-Nitropropane-dioxygenase activity was assayed with 10 mm 2-nitropropane (anionic form) as a substrate. The reaction was carried out at 30 °C in a final volume of 1 ml of Britton-Robinson's buffer (pH 6.5) (a mixture of 16 mm phosphoric acid, 16 mm acetic acid, and 16 mm boric acid, whose pH is adjusted with NaOH), and the amount of nitrite was determined with sulfanilamide and N-(1-naphthyl)ethylenediamine by measuring the absorbance at 540 nm (19Kido T. Soda K. Arch. Biochem. Biophys. 1984; 234: 468-475Crossref PubMed Scopus (27) Google Scholar). One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 μmol of nitrite per minute. Protein concentrations were measured with a Bio-Rad protein and were using the The were by DNA were by the at °C by of the protein in a buffer of 50 mm Tris-HCl at pH mm and 100 mm sodium and the To we used a of m at pH at pH and to of mm × mm × mm a To of the protein with 2-nitropropane mm 2-nitropropane in 50 mm Tris-HCl, pH and 100 mm sodium was with the protein in a volume in an of 2-nitropropane the enzyme. The protein with 2-nitropropane was incubated for 30 at °C of as the a The SeMet-substituted protein was crystallized to for the except for the presence of 10 mm dithiothreitol in the protein The of the SeMet-substituted to of × × mm a Data 1 the of A crystal of the SeMet-substituted protein was into a for a and then in the at 100 K. The of m at pH at pH and were at 100 at using an at the of The crystal was through a of with a per The were and using the Z. W. Scopus Google Scholar). The SeMet-substituted crystal to the with unit cell of a A is in the with the crystal volume per protein of 3The abbreviations used are: SeMet, selenomethionine; MAD, multiwavelength anomalous diffraction; PDB, Protein Data Bank; r.m.s., root mean square. and the of of the protein as a ternary complex with both FMN and were as of the protein as a binary complex with FMN were using a at the of The were and using the Z. W. Scopus Google and and cell a in are for the in are for the in are for the in are for the is the of is the all and is the of in for the is the of is the all and is the of in for the is the of is the all and is the of in for the is the of is the all and is the of in for the of of is the phase and is the phase for in to the in to the in to the in to the of is for a of which were not used for structure and was for the for the are for the binary complex with FMN and for the ternary complex with FMN and 2-nitropropane of protein of of from in are for the is the of is the all and is the of in for the of is the phase and is the phase in to the is for a of which were not used for structure and was for the for the are for the binary complex with FMN and for the ternary complex with FMN and 2-nitropropane in a and are in of the expected in of the recombinant except the at the terminus of the were with the J. D. PubMed Scopus Google and the were used to the with the D. 2003; PubMed Scopus Google Scholar). The was of to by D. 2003; PubMed Scopus Google an that for of the of the with of the sequence was done using the M. A. 1991; PubMed Scopus Google Scholar). The was with the P. J. M. N.S. T. D. 1998; PubMed Scopus Google the of the were as the for the of Nature. 1992; PubMed Scopus Google Scholar). of and were this was used to of the enzyme as a binary complex with FMN and as a ternary complex with both FMN and 2-nitropropane. are in of the have as by the J. Appl. Google Scholar). Protein Expression and 2-nitropropane dioxygenase from P. aeruginosa was expressed in E. coli as a with the tag of the expressed protein was in the The was of the enzyme per of This was to a considerable of the enzyme that during the gel the protein and by gel filtration we the The recombinant enzyme has a molecular of the tag as by This that P. aeruginosa 2-nitropropane dioxygenase is in the 2-nitropropane dioxygenase from N. crassa (16Francis K. Russell B. Gadda G. J. Biol. Chem. 2005; 280: 5195-5204Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The of the recombinant enzyme in the and nm that of the containing the flavin V. Biochem. 2000; PubMed Google Scholar). the presence of non-covalently bound FMN and We have also found that the recombinant enzyme the enzyme 2-nitropropane dioxygenase compounds the enzyme not the enzyme activity are similar to for N. crassa 2-nitropropane dioxygenase (14Gorlatova N. Tchorzewski M. Kurihara T. Soda K. Esaki N. Appl. Environ. Microbiol. 1998; 64: 1029-1033Crossref PubMed Google Scholar). acid, a of from G. A. J. Enzyme 2001; PubMed Scopus Google not the P. aeruginosa 2-nitropropane dioxygenase activity We also the substrate 2-nitropropane bound with FMN in the by the of we that the PA1024 gene product is a 2-nitropropane of FMN and 2-nitropropane in the A, binding of FMN in the binary complex A is the of of the binding of FMN and 2-nitropropane in the ternary complex showing the P. aeruginosa 2-nitropropane dioxygenase and FMN and 2-nitropropane in the ternary complex of P. aeruginosa 2-nitropropane dioxygenase bound with FMN and 2-nitropropane. In this of the the substrate and the cofactor are the have determined the crystal structure of P. aeruginosa 2-nitropropane dioxygenase using The of the binary complex with FMN 1 and was to and of and respectively The binary complex Data for and 1 FMN in the We have determined the structure of the ternary complex with FMN and 2-nitropropane. The ternary complex was to and of and respectively The for amino acid 1 FMN and 1 2-nitropropane two and two of 2-nitropropane as as the eight-residue are from both because is no The of binary and ternary are highly similar to with a root mean of for which on a and shows the This that is no substrate The enzyme has of × × 60 structure of two a and and an inserted A and The two are by two The is into the with a of by and A and The the of the on the of the The inserted and the of the The of the is of the and its out from the of the The inserted of two and the structure The is at the the two with the and A and This the FMN binding on the of the in the A of the inserted the bound FMN A and structural similarity L. C. J. Mol. Biol. PubMed Scopus Google Scholar) with the inserted not any similar The is with the protein an of for 37 a of and a sequence identity of This that the inserted has a The L. C. J. Mol. Biol. PubMed Scopus Google Scholar) with the many similar as one can from its The is with the from an of for a of and a sequence identity of P. C.C. PubMed Scopus Google Scholar). The is with an of for a of and a sequence identity of of FMN and we have not FMN in the one of FMN is non-covalently bound in the of the recombinant enzyme and FMN is the the two on the of the in the A and The of the is not the of the to the is from the protein A and The of the FMN binding is by the The is by the following of the and The inserted to the FMN binding A of the inserted the part of the and to the binding of the substrate 2-nitropropane and The of FMN the of and the two and from and The and the binding that is also by members of the and family of N. J. Mol. Biol. PubMed Scopus Google Scholar). In 2-nitropropane the also to with the to its is to the but its from the The of the bound FMN no significant from the The of the is to the of and This is in with the previous that the of flavin is in with a A. Biochem. 2000; Full Text Full Text PDF Scopus Google Scholar). The of is to the and of In the of FMN is to the of and two The of the of FMN is to the carbonyl of and the of The of the is to the of and We have also determined the structure of P. aeruginosa 2-nitropropane dioxygenase as a ternary complex with both FMN and the neutral of the substrate 2-nitropropane. The enzyme is and thus the reaction with the neutral of 2-nitropropane not in the crystal to an the crystal was for a The of 2-nitropropane not the nitro from the isopropyl In one of the the of and one the of the substrate with the nitro a in the of the substrate. Therefore, we the of 2-nitropropane. The substrate is the protein on the of the of FMN and and is from the This of substrate is also observed in flavin-dependent and A. Biochem. 2000; Full Text Full Text PDF Scopus Google Scholar). In the binary complex with FMN a is at the substrate The highly conserved and of the of the flavin In both of the binary and ternary a at is in the of the its is defined by the This is part of a conserved sequence and is to the conserved that with the bound FMN and not to be in binding FMN the substrate and The of is for the to a the and of that the PA1024 gene product has significant levels of amino acid sequence similarity to 2-nitropropane dioxygenases from various sources. of 2-nitropropane dioxygenases identifies at of conserved amino acid They are and for any amino acid, is a and the that are conserved the are in The first to the and are in binding the cofactor and the substrate. They that the binding for FMN and by the in The and that to are as the The highly conserved His152 by an the in which we as the likely candidate for the catalytic of the to the inserted and is in with the highly conserved of the to structural of the 2-Nitropropane dioxygenases from the fungus N. crassa and the yeast W. mrakii (H. mrakii) sequence of and to P. aeruginosa 2-nitropropane respectively Despite to levels of sequence the and that the and which we as the catalytic of the are conserved in the and yeast enzymes of the previous study on the catalytic mechanism of 2-nitropropane dioxygenase from N. crassa that the oxidation of nitroalkanes both their neutral and anionic by 2-nitropropane dioxygenase by an oxidase-style mechanism, in which the oxidation of the substrate and the oxidation of the enzyme-bound flavin in two independent (16Francis K. Russell B. Gadda G. J. Biol. Chem. 2005; 280: 5195-5204Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). neutral nitroalkanes as the is by from the of the substrate to enzyme-bound which are then following the same catalytic pathway for the anionic of the substrate (16Francis K. Russell B. Gadda G. J. Biol. Chem. 2005; 280: 5195-5204Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). of the neutral nitroalkanes by 2-nitropropane dioxygenase from N. crassa a catalytic with of which oxidation of the neutral substrates by the from the substrate (16Francis K. Russell B. Gadda G. J. Biol. Chem. 2005; 280: 5195-5204Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). A similar for a catalytic that the from the substrate was previously in from a fungus F. a of the family of the nitroalkane-oxidizing enzymes, the catalytic was as with J. Chem. 2003; PubMed Scopus Google Scholar). However, the identity of the catalytic in 2-nitropropane dioxygenases has to the of sequence similarity 2-nitropropane dioxygenase and and the of structural information on 2-nitropropane dioxygenase. of P. aeruginosa 2-nitropropane dioxygenase that His152 and are in of 2-nitropropane on the of the of FMN in the and two for the catalytic base, we that His152 is likely to as the catalytic It to the highly conserved sequence and is conserved and 2-nitropropane dioxygenases from N. crassa and W. mrakii (H. mrakii) We that the in N. crassa and in W. mrakii (H. mrakii), the of the catalytic are many in which a similar as a catalytic For example, of from Pseudomonas as the that the to a in the of the reaction B. PubMed Scopus (41) Google Scholar). His152 of P. aeruginosa 2-nitropropane dioxygenase is in such a that its with the of 2-nitropropane and is to the of the neutral substrate in the of the reaction to a To the proposed of the conserved His152 in we performed a However, the was expressed in in E. of P. aeruginosa 2-nitropropane dioxygenase to a of the inserted The of the nitro of 2-nitropropane via a and This the of the its by To the of we with the enzyme we overexpressed in E. To we have determined the first crystal structure of 2-nitropropane dioxygenase from P. The cofactor FMN is bound non-covalently in the and the structure suggests that conserved His152 likely as the catalytic that oxidation of neutral substrates by a from the We the at of and and of the for during with