Structural Basis of 5-Nitroimidazole Antibiotic Resistance
2004; Elsevier BV; Volume: 279; Issue: 53 Linguagem: Inglês
10.1074/jbc.m408044200
ISSN1083-351X
AutoresHanna‐Kirsti S. Leiros, S. Kozielski-Stuhrmann, U. Kapp, Laurent Terradot, Gordon A. Leonard, Seán McSweeney,
Tópico(s)Enzyme Structure and Function
Resumo5-Nitroimidazole-based antibiotics are compounds extensively used for treating infections in humans and animals caused by several important pathogens. They are administered as prodrugs, and their activation depends upon an anaerobic 1-electron reduction of the nitro group by a reduction pathway in the cells. Bacterial resistance toward these drugs is thought to be caused by decreased drug uptake and/or an altered reduction efficiency. One class of resistant strains, identified in Bacteroides, has been shown to carry Nim genes (NimA, -B, -C, -D, and -E), which encode for reductases that convert the nitro group on the antibiotic into a non-bactericidal amine. In this paper, we have described the crystal structure of NimA from Deinococcus radiodurans (drNimA) at 1.6 Å resolution. We have shown that drNimA is a homodimer in which each monomer adopts a β-barrel fold. We have identified the catalytically important His-71 along with the cofactor pyruvate and antibiotic binding sites, all of which are found at the monomer-monomer interface. We have reported three additional crystal structures of drNimA, one in which the antibiotic metronidazole is bound to the protein, one with pyruvate covalently bound to His-71, and one with lactate covalently bound to His-71. Based on these structures, a reaction mechanism has been proposed in which the 2-electron reduction of the antibiotic prevents accumulation of the toxic nitro radical. This mechanism suggests that Nim proteins form a new class of reductases, conferring resistance against 5-nitroimidazole-based antibiotics. 5-Nitroimidazole-based antibiotics are compounds extensively used for treating infections in humans and animals caused by several important pathogens. They are administered as prodrugs, and their activation depends upon an anaerobic 1-electron reduction of the nitro group by a reduction pathway in the cells. Bacterial resistance toward these drugs is thought to be caused by decreased drug uptake and/or an altered reduction efficiency. One class of resistant strains, identified in Bacteroides, has been shown to carry Nim genes (NimA, -B, -C, -D, and -E), which encode for reductases that convert the nitro group on the antibiotic into a non-bactericidal amine. In this paper, we have described the crystal structure of NimA from Deinococcus radiodurans (drNimA) at 1.6 Å resolution. We have shown that drNimA is a homodimer in which each monomer adopts a β-barrel fold. We have identified the catalytically important His-71 along with the cofactor pyruvate and antibiotic binding sites, all of which are found at the monomer-monomer interface. We have reported three additional crystal structures of drNimA, one in which the antibiotic metronidazole is bound to the protein, one with pyruvate covalently bound to His-71, and one with lactate covalently bound to His-71. Based on these structures, a reaction mechanism has been proposed in which the 2-electron reduction of the antibiotic prevents accumulation of the toxic nitro radical. This mechanism suggests that Nim proteins form a new class of reductases, conferring resistance against 5-nitroimidazole-based antibiotics. Antibiotic resistance is an increasing problem throughout the developed world, and knowledge about different resistance mechanisms is important for efficient treatment of bacterial infections. One important class of antibiotics, the 5-nitroimidazole (5-Ni) 1The abbreviations used are: 5-Ni, 5-nitroimidazole; drNimA, 5-nitroimidazole antibiotic-resistant protein from D. radiodurans; bfNimA, NimA from B. fragilis; MTR, 2-methyl-5-nitroimidazole-1-ethanol (metronidazole); DMZ, 1,2-dimethyl-5-nitroimidazole (dimetridazole); TNZ, 2-ethylsulfonyl 1-ethyl 2-methyl 5-nitroimidazole (tinidazole); PFOR, pyruvate-ferredoxin oxidoreductase; FMN, flavin mononucleotide; MES, 4-morpholineethanesulfonic acid; PNPO, pyridoxine 5′-phosphate oxidase; Pyr, pyruvate, Lac, lactate; r.m.s.d., root mean square deviation. drug derivatives, includes metronidazole (MTR), dimetridazole (DMZ), and tinidazole (TNZ). MTR is extensively used in the treatment of anaerobic infections caused by Trichomonas vaginalis, Entamoeba histolytica, Enterococcus species, Giardia lamblia, Clostridium species, and Bacteroides (1Edwards D.I. J. Antimicrob. Chemother. 1993; 31: 9-20Crossref PubMed Scopus (480) Google Scholar, 2Edwards D.I. J. Antimicrob. Chemother. 1993; 31: 201-210Crossref PubMed Scopus (123) Google Scholar, 3Rafii F. Wynne R. Heinze T.M. Paine D.D. FEMS Microbiol. Lett. 2003; 225: 195-200Crossref PubMed Scopus (34) Google Scholar, 4Fang H. Edlund C. Hedberg M. Nord C.E. Int. J. Antimicrob. Agents. 2002; 19: 361-370Crossref PubMed Scopus (41) Google Scholar) and is also a critical ingredient of modern multidrug therapies for Helicobacter pylori eradication regimes used to control ulcers (5Houben M.H. van de Beek D. Hensen E.F. Craen A.J. Rauws E.A. Tytgat G.N. Aliment. Pharmacol. Ther. 1999; 13: 1047-1055Crossref PubMed Scopus (305) Google Scholar). The mode of action for the 5-Ni antibiotics, as illustrated in Fig. 1, has been shown to be similar in different pathogens (6Kulda J. Int. J. Parasitol. 1999; 29: 199-212Crossref PubMed Scopus (143) Google Scholar, 7Lockerby D.L. Rabin H.R. Laishley E.J. Antimicrob. 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Chemother. 1993; 31: 201-210Crossref PubMed Scopus (123) Google Scholar, 10Declerck P.J. De Ranter C.J. Biochem. Pharmacol. 1986; 35: 59-61Crossref PubMed Scopus (40) Google Scholar, 11Declerck P.J. de Ranter C.J. Volckaert G. FEBS Lett. 1983; 164: 145-148Crossref PubMed Scopus (21) Google Scholar), and damage to other vital cell systems is also possible (6Kulda J. Int. J. Parasitol. 1999; 29: 199-212Crossref PubMed Scopus (143) Google Scholar). The success of such drugs depends on the reductive activation of the nitro group on the 5-Ni drug, which is controlled by the redox system of the target cell. As a consequence, species with altered, absent, or elevated redox potential pathways are resistant to 5-Ni drugs (see Ref. 12Kwon D.H. El-Zaatari F.A. Kato M. Osato M.S. Reddy R. Yamaoka Y. Graham D.Y. Antimicrob. Agents Chemother. 2000; 44: 2133-2142Crossref PubMed Scopus (107) Google Scholar and references therein). For H. pylori, the most convincing data regarding MTR resistance relate to inactivation of the RdxA gene, which encodes an oxygen-insensitive NADPH nitroreductase (13Marais A. Bilardi C. Cantet F. Mendz G.L. Megraud F. Res. Microbiol. 2003; 154: 137-144Crossref PubMed Scopus (76) Google Scholar). Still, resistance has been found in H. pylori strains with an intact RdxA gene (14Jenks P.J. Ferrero R.L. Labigne A. J. Antimicrob. Chemother. 1999; 43: 753-758Crossref PubMed Scopus (93) Google Scholar). In T. vaginalis, a reduced amount of available ferredoxin as an electron acceptor/donor is thought to be responsible for drug resistance (15Quon D.V. d'Oliveira C.E. Johnson P.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4402-4406Crossref PubMed Scopus (83) Google Scholar), but strains with knock-out ferredoxin genes are not resistant under aerobic or anaerobic conditions (16Land K.M. Delgadillo-Correa M.G. Tachezy J. Vanacova S. Hsieh C.L. Sutak R. Johnson P.J. Mol. Microbiol. 2004; 51: 115-122Crossref PubMed Scopus (47) Google Scholar). Therefore, it is likely that multiple pathways lead to both activation and resistance of the MTR and other 5-Ni drugs. The 5-Ni resistance of some of Bacteroides fragilis strains was shown to be mediated by specific genes, named Nim, located either on the chromosome (NimB) or on small mobilizable plasmids, e.g. pIP417 (NimA), pIP419 (NimC), and pIP421 (NimD) (17Breuil J. Dublanchet A. Truffaut N. Sebald M. Plasmid. 1989; 21: 151-154Crossref PubMed Scopus (62) Google Scholar, 18Haggoud A. Reysset G. Azeddoug H. Sebald M. Antimicrob. Agents Chemother. 1994; 38: 1047-1051Crossref PubMed Scopus (71) Google Scholar, 19Haggoud A. Reysset G. Sebald M. FEMS Microbiol. Lett. 1992; 74: 1-5Crossref PubMed Google Scholar, 20Trinh S. Haggoud A. Reysset G. Sebald M. Microbiology. 1995; 141: 927-935Crossref PubMed Scopus (56) Google Scholar). A fifth Nim gene, NimE, was discovered that confers resistance to high MTR concentrations in strains from Bacteroides thetaiotaomicron, B. fragilis, and Bacteroides ovatus (21Stubbs S.L. Brazier J.S. Talbot P.R. Duerden B.I. J. Clin. Microbiol. 2000; 38: 3209-3213Crossref PubMed Google Scholar). The enzymatic activity of the Nim gene products was deduced by comparing the metabolism of a 5-Ni-susceptible strain with the same strain harboring a plasmid containing the NimA sequence from B. fragilis (bfNimA) (22Carlier J.P. Sellier N. Rager M.N. Reysset G. Antimicrob. Agents Chemother. 1997; 41: 1495-1499Crossref PubMed Google Scholar). In the sensitive strain, the classic reduction of DMZ to its nitro radical anion was observed in agreement with the general scheme (see Fig. 1). However, in the resistant strain, DMZ was reduced to its amine derivative (R-NH2) through a low redox potential reaction. The addition of pyruvate, which can act as an electron donor through the PFOR complex (Fig. 1), increased the drug uptake (22Carlier J.P. Sellier N. Rager M.N. Reysset G. Antimicrob. Agents Chemother. 1997; 41: 1495-1499Crossref PubMed Google Scholar). Hence, it was proposed that Nim proteins are 5-Ni reductases, which possibly use ferredoxin as the electron donor (22Carlier J.P. Sellier N. Rager M.N. Reysset G. Antimicrob. Agents Chemother. 1997; 41: 1495-1499Crossref PubMed Google Scholar). Following the increasing availability of new bacterial genome sequences, it emerges that Nim homologues are present in other genera of bacteria, including Deinococcus radiodurans, B. fragilis, Helicobacter hepaticus, Clostridium sp., Salmonella typhi, Streptomyces avermitilis, as well as in Archaea Methanosarcina sp. (Fig. 2). Although the underlying physiological function of the Nim homologues in these organisms is unknown, it seems likely that the Nim gene family is ancient and widespread. More importantly, because of its unstable nature on mobilizable plasmids, Nim gene-based resistance poses a real threat to the existing applications of 5-Ni drugs. Indeed, a recent study identified seven Bacteroides-resistant strains with minimum inhibitory concentrations of >32 μg/ml, all containing Nim genes (23Jamal W.Y. Rotimi V.O. Brazier J.S. Johny M. Wetieh W.M. Duerden B.I. Med. Princ. Pract. 2004; 13: 147-152Crossref PubMed Scopus (11) Google Scholar). In this paper, we present the first crystal structure of a Nim enzyme, the NimA from D. radiodurans (drNimA, DR0842) at 1.6 Å resolution as well as its complexes with the MTR antibiotic, covalently bound pyruvate and covalently bound lactate. drNimA shares 28% sequence identity and 54% sequence homology with bfNimA, and should thus be representative of the Nim family as a whole. Taken together with previous studies on NimA from B. fragilis and comparison with other closely related enzymes, our observations suggest that Nim proteins do indeed function as reductases. We propose a mechanism for the reduction of 5-Ni compounds by Nim enzymes that leads to generation of non-toxic derivatives and confers resistance against these antibiotics. Cloning, Protein Expression, and Purification of DR0842—The gene DR0842 (drNimA, 21.9 kDa), was cloned from genomic D. radiodurans DNA into the Gateway destination vector pDEST17 (Invitrogen) by the company Protein'eXpert SA, Grenoble, France. The correct sequence with an amino-terminal hexahistidine tag (sequence MSYYHHHHHHLESTSLYKKAG) has been confirmed by DNA sequencing. E. coli BL21(DE3)pLysS cells (Novagen) transformed with pDEST17-drNimA were grown at 37 °C in rich broth 2×YT medium (16 g/liter bacto-tryptone, 10 g/liter bacto-yeast extract, 10 g/liter NaCl) with 100 mg/liter ampicillin and 34 mg/liter chloramphenicol, and at A600 of 0.5, the cells were induced with 1 mm isopropyl-β-d-thiogalactopyranoside for 4 h at 37 °C. The harvested cells were resuspended in lysis buffer (20 mm Tris-HCl, pH 7.2, 500 mm NaCl, 10 mm imidazole) supplemented with DNaseI, Lysozyme, and Complete EDTA-free Protease Inhibitor Mixture (Roche) and lysed by sonication, and the soluble lysate was applied to nickel-nitrilotriacetic acid resin (Qiagen). The protein was eluted with a linear gradient of imidazole at a concentration of from 10 to 500 mm. Fractions containing drNimA were de-salted (HiTrap desalting column, Pharmacia Corporation) and loaded onto a MonoQ column (Amersham Bioscience) and further eluted with a NaCl gradient (0-1 m) in which drNimA eluted at around 0.1 m NaCl. Fractions with drNimA were pooled and further purified by analytical gel filtration (Superdex 200, Pharmacia Corporation) in a buffer consisting of 10 mm Tris-HCl, pH 7.5, 1 mm dithiothreitol, 1 mm EDTA. The peak fractions were concentrated to 20 mg/ml. Electrospray mass spectrometry of the purified drNimA confirmed the molecular mass of ∼24 kDa (drNimA 21.9 kDa + His-tag 2.5 kDa). Oligomeric State and Cross-linking—An additional gel filtration run (in 25 mm Tris-HCl, pH 8.0, 100 mm NaCl) was also carried out with drNimA and the standard molecular mass markers, albumin (67 kDa) and chymotrypsin (25 kDa), to correlate the elution volume of drNimA to the molecular mass of the protein in solution. One run with only the molecular mass marker, ovalbumin (43 kDa), was also performed. All four proteins were at concentrations of ∼1 mg/ml. Another analytical gel filtration run (Superdex 200 (Pharmacia Corporation) in 50 mm Hepes, pH 7.5, and 1 mm EDTA) with the pure drNimA protein was performed to change the buffer from Tris to Hepes, and this elution curve is given in Fig. 4a. The main peak from this run was used for the cross-linking experiments. A series of 0, 0.25, 0.50, 1.0, and 5.0 mm cross-linker, ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester) (Sigma) in Me2SO was added to the protein and left for 20 min on the bench, and then Tris was added to a final concentration of 60 mm. The series was analyzed on a 12% SDS-PAGE gel (see Fig. 4b). Crystallization, Soaking, Data Collection, and Structure Solution—Initial screening for suitable crystallization conditions was carried out by the hanging drop method using standard commercial screening solutions. The final crystallization conditions at 4 °C had 4-μl hanging drops consisting of a 1:1 mixture of protein (6 mg/ml) and reservoir solution with 0.65-0.9 m sodium acetate and 0.1 m sodium cacodylate, citrate, or MES buffered at pH 5.5-6.0. The crystals grew as rosettes with plate-like "fingers," which were cracked, and the resulting plates with an approximate size of 100 × 30 × 5 μm3 were used for data collection. For structure solution purposes, a mercury derivative (drNimA-Hg) was prepared by soaking the crystal in a solution with 1 mm ethyl mercury thiosalisylate, 0.2 m sodium acetate, 0.1 m sodium cacodylate, pH 6.0, and 30% polyethylene glycol 4000 at 4 °C for 20 min. The complex structures described in this paper were prepared by soaking the native crystals in solution containing 0.2 m sodium acetate, 0.1 m sodium cacodylate, pH 6.0, and 30% polyethylene glycol 4000 and 1, 5, or 10 mm MTR or TNZ (both from Sigma) at 4 °C. As will be seen, soaking for 2 h in 10 mm MTR produced a structure with drNimA in complex with MTR (drNimA-MTR), whereas soaking for 20 h in 1 mm TNZ yielded a complex with covalently bound pyruvate (drNimA-Pyr), and soaking for 23 h in 1 mm MTR produced a complex with drNimA and covalently bound lactate (drNimA-Lac). All diffraction data were collected at the European Synchrotron Radiation Facility, Grenoble, France, using crystals cooled to 100 K. Cryo-protection for the native crystals was effected by leaving the crystals for ∼20 s in a solution with 0.2 m sodium acetate, 0.1 m sodium cacodylate, pH 6.0, and 30% polyethylene glycol 4000. Crystals of the mercury derivative and of the various complexes could all be frozen straight from their soaking solutions. The crystals of drNimA (including the substrate soaks) showed great sensitivity to ambient temperatures; therefore all manipulations described above and flash freezing in liquid nitrogen were preformed in a cold room maintained at 4 °C. All crystals belong to the space group C2, and the unit cell dimensions for the native crystal were a = 99.86 Å, b = 38.95 Å, c = 59.81 Å, and β = 114.25° (see Table I for further details). All data were integrated using MOSFLM, scaled with SCALA, and structure factors obtained using TRUNCATE software (24Collaborative Computational Project No 4 Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar).Table IStatistics from the data collectionsX-ray statisticsdrNimA-Hg (EMTS)drNimA (Native)drNimA-MTRdrNimA-PyrdrNimA-LacBeamlineID14-EH2ID14-EH1ID14-EH4ID14-EH2ID14-EH4Space groupC2C2C2C2C2PDB entry1w3o1w3r1w3p1w3qUnit cella = 100.09a = 99.86a = 99.59a = 99.94a = 99.80b = 39.15b = 38.95b = 39.17b = 38.81b = 38.77c = 59.95c = 59.81c = 59.84c = 60.06c = 60.00β = 114.39°β = 114.25°β = 114.09°β = 114.11°β = 114.04°Resolution (Å) (highest bin)20-2.10 (2.21-2.10)20-1.60 (1.69-1.60)30-1.90 (2.00-1.90)30-1.80 (1.90-1.80)30-1.88 (1.98-1.88)Wavelength (Å)0.9330.9340.9390.9330.939No. of unique reflections12,56726,05616,84619,53017,262Multiplicity8.1 (8.2)3.7 (3.7)4.3 (4.4)2.7 (2.7)2.5 (2.5)Completeness (%)99.9 (100.0)99.9 (100.0)99.9 (99.9)99.0 (99.5)99.8 (100.0)Intensity (I/σ1)5.3 (1.5)7.6 (1.5)3.4 (1.7)3.9 (0.8)6.9 (1.5)Mean (〈I〉/〈σI〉)15.4 (4.3)11.2 (2.8)9.3 (2.9)9.8 (1.7)9.9 (2.2)Rsym (%)aRsym=(∑h∑i|Ii(h)−〈I(h)〉|)/(∑h∑II(h)), where Ii(h) is the ith measurement of reflection h and 〈I(h)〉 is the weighted mean of all measurements of h.12.8 (48.2)7.4 (44.8)14.2 (37.9)8.90 (69.0)8.1 (46.6)Ranom (%)bRanom=∑(|〈I+〉−〈I−〉|)/(∑(〈I+〉+〈I−〉)), where 〈I〉 is the mean intensity of the reflection.5.7 (18.4)FOMSIRAScFOMSIRAS = figure of merit after SIRAS phasing.0.45 (to 2.1 Å)FOMSFdFOMSF = figure of merit after solvent flattening.0.46 (to 1.6 Å) 0.69 (to 2.3 Å)a Rsym=(∑h∑i|Ii(h)−〈I(h)〉|)/(∑h∑II(h)), where Ii(h) is the ith measurement of reflection h and 〈I(h)〉 is the weighted mean of all measurements of h.b Ranom=∑(|〈I+〉−〈I−〉|)/(∑(〈I+〉+〈I−〉)), where 〈I〉 is the mean intensity of the reflection.c FOMSIRAS = figure of merit after SIRAS phasing.d FOMSF = figure of merit after solvent flattening. Open table in a new tab The structure of drNimA was elucidated using the single isomorphous difference with anomalous scattering technique, with initial experimental phases based on the single ethyl mercury thiosalisylate derivative obtained to 2.1 Å using the software program SOLVE (25Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). Phase improvement and extension to 1.6 Å resolution were carried out with RESOLVE (26Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 1688-1701Crossref PubMed Scopus (31) Google Scholar), and ARP/wARP software (27Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2565) Google Scholar) was then used to produce an initial model for drNimA (192 of the final 224 residues). There is one monomer in the crystallographic asymmetric unit resulting in a solvent content of 43% and a Matthews coefficient of 2.2 Å3/Da. Phases were improved by iterative cycles of refinement in the REFMAC program (28Murshudov G.N. Vagin A.A. Lebedev A. Wilson K.S. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 247-255Crossref PubMed Scopus (1010) Google Scholar) interspersed with rounds of manual rebuilding in O software (29Jones T.A. Zou J-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar), during which solvent molecules were incorporated. The structures of the drNimA complexes described here were obtained by using the final model of the native structure (drNimA) stripped for solvent molecules as the starting point for the refinement, and model improvement was carried out as for the native structure. Full details of the data collections and the results of all refinements are given in Tables I and II.Table IIRefinement statistics for the presented structuresNative drNimAdrNimA-MTRdrNimA-PyrdrNimA-LacR-factor (all reflections) (%)a∑h||Fobs|−|Fcalc||/∑h|Fobs|, where |Fobs| and |Fcalc| are observed and calculated structure factor amplitudes for all reflections (R-factor) and the reflections applied in the test Rfree set (reflection not used in the structure refinement), respectively.16.4519.2018.7317.23Rfree (%)a∑h||Fobs|−|Fcalc||/∑h|Fobs|, where |Fobs| and |Fcalc| are observed and calculated structure factor amplitudes for all reflections (R-factor) and the reflections applied in the test Rfree set (reflection not used in the structure refinement), respectively.21.1125.5523.1122.36No. of protein atoms1637163716371637No. of water molecules333292293270No. of other molecules1 acetate1 acetate1 acetate1 acetate1 pyruvate1 pyruvate1 His-pyruvate1 His-lactate1 MTRR.m.s.d. bond lengths (Å)0.0190.0140.0140.014R.m.s.d. bond angles (°)1.7091.4761.4511.514Average B-factor (Å2)All atoms15.1020.8423.5726.58Protein (Res. 2-195)12.6118.5521.3524.63Water molecules25.1129.1833.6434.42Acetate13.0624.4423.2324.52Pyruvate/His-Pyr/His-Lac28.9539.0925.0426.41MTR61.16Ramachandran plot: Most favored region (%)91.991.390.889.6Additionally allowed regions (%)8.18.79.210.4a ∑h||Fobs|−|Fcalc||/∑h|Fobs|, where |Fobs| and |Fcalc| are observed and calculated structure factor amplitudes for all reflections (R-factor) and the reflections applied in the test Rfree set (reflection not used in the structure refinement), respectively. Open table in a new tab Structure, Fold, and Dimerization of drNimA—The crystal structure of drNimA comprises one monomer in the asymmetric unit, and the final model of the native structure (drNimA) comprises 10 of the 21 residues in the amino-terminal His-tag, including the six histidines, 194 of 195 residues of the protein itself, one acetate ion, one pyruvate molecule, and 334 water molecules. The approximate dimensions of the monomer (Fig. 3a) are 65 × 45 × 25 Å3. The structure consists of a central six-stranded anti-parallel β-barrel with strand order β1, β2, β3, β6, β5, β4. The β-barrel is non-symmetric with β-strands β4, β5, and β6 elongating the barrel toward both the amino and carboxyl termini of the protein. These three extended strands are perpendicular to the helix α1 (from Asp-24 to Arg-33) that locks the bottom of the β-barrel as shown in Fig. 3a. Opposite the expanded strands, two helices (α2, α3) flank the β-barrel. Analytical gel filtration and cross-linking experiments clearly indicate that drNimA is a homodimer (Fig. 4). This homodimer is, in the crystals, formed by the crystallographic 2-fold axis. When the dimer is being formed, it buries a surface area of 2314 Å2 that is 18% of the area in a monomer. In the dimer, the long β-strands (β4, β5, β6) of one monomer grip onto the β-barrel of the other monomer forming a β-propeller with ten strands (Fig. 3, c and e) with approximate dimensions of 75 × 47 × 43 Å3. The two drNimA monomers are held together by 10 hydrogen bonds, including a salt bridge between Arg-38 and the carboxyl group of Glu-91, some aromatic interactions, and several water-mediated interactions at the dimer interface. Native Structure—During the refinement of the drNimA structure, a flat, X-shaped portion of difference electron density was observed in a solvent-exposed pocket on the monomer-monomer interface (Fig. 5a). This density is at hydrogen-binding distance to the absolutely conserved residue His-71 (Fig. 2). To form an interpretation of this electron density, several possible molecules were tested for suitability. Only molecules satisfying the constraints of size and shape placed by the difference electron density, and also conforming to the chemical environment available, were considered. Ultimately, the most satisfactory explanation of the residual difference density was obtained by the incorporation of a pyruvate moiety into the model. The assignment of a pyruvate molecule here seems to be reasonable, because all three oxygen atoms are involved in hydrogen binding networks (Fig. 5b), and the hydrophobic methyl group (of the pyruvate) is facing Phe-140 from one molecule and Phe-98′ from the second monomer (Fig. 5b, where the "′" implies residues from the second monomer in the homodimer). The refined pyruvate moiety fits within the observed electron density (Fig. 6a), it has reasonable bond lengths and bonds angles, and no difference density was observed when the structure refinement was completed.Fig. 6Electron density maps. A, the final σA-weighted 2Fo - Fc electron density map (1.2σ) of the native drNimA structure with only the pyruvate in the active site. B, the active site of the complex with pyruvate and the MTR antibiotic (drNimA-MTR). The 2Fo - Fc map (1.0σ) is contoured together with some inter-atomic distances. The orientation is slightly different from panel A, but the sizes are identical. C, shown are the soaks, which resulted in a covalently bound pyruvate (drNimA-Pyr) with the corresponding 2Fo - Fc map (1.2σ). D, shown is the covalently bound lactate molecule (drNimA-Lac) with its 2Fo - Fc map (1.1σ). In panel D, the His-71-Pyr residue of the drNimA-Pyr structure is included as solid black bonds. The orientation of panels B-D are identical, but the sizes are bigger in panels C and D to focus on the covalent links. All figures have Fo - Fc maps at +4σ (green) and -4σ (red) and were made using BobScript (42Esnouf R.M. J. Mol. Graph. 1997; 15: 132-134Crossref Scopus (1795) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the final native structure, the pyruvate is located between the β2 and β3 strands, and O-1 in the carboxyl group is 2.38 Å away from His-71 Nϵ-2. The three oxygen atoms of the pyruvate are hydrogen bound to the four water molecules W-1, W-2, W-3, and W-5. The amino acids Val-139 and Leu-107′, along with Phe-140 and Phe-98′, also contribute to the binding site (Figs. 3f and 6a). Comparison of Sequence and Structural Homologues—To search for sequence and structural homologues, the drNimA sequence and coordinates were used with the BLAST (30Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar) and DALI servers (31Holm L. Sander C. Nucleic Acids Res. 1997; 25: 231-234Crossref PubMed Scopus (361) Google Scholar). BLAST identified a number of sequence homologues in the data base, which included sequences from the Clostridium species, Bacteroides species, H. hepaticus and S. typhi, and Archaea (Methanosarcina mazei). The sequence identity of drNimA toward the sequences included in Fig. 2 is 14-23%, and the homology is 46-55%. Two sequence motifs were found in the Conserved Domain Database (32Marchler-Bauer A. Anderson J.B. DeWeese-Scott C. Fedorova N.D. Geer L.Y. He S. Hurwitz D.I. Jackson J.D. Jacobs A.R. Lanczycki C.J. Liebert C.A. Liu C. Madej T. Marchler G.H. Mazumder R. Nikolskaya A.N. Panchenko A.R. Rao B.S. Shoemaker B.A. Simonyan V. Song J.S. Thiessen P.A. Vasudevan S. Wang Y. Yamashita R.A. Yin J.J. Bryant S.H Nucleic Acids Res. 2003; 31: 383-387Crossref PubMed Scopus (650) Google Scholar): flavin mononucleotide (FMN) binding and pyridoxamine 5′-phosphate oxidase. The DALI server confirmed these results by identifying other proteins with similar β-barrel fold as drNimA, which are all FMN-binding proteins. They include the human pyridoxine 5′-phosphate oxidase (PNPO) (Protein Data Bank (PDB) code 1nrg, Z-score 4.0, root mean square deviation 2.15 Å over 97 Cα atoms), the FMN-binding protein from Desulfovibrio vulgaris (PDB code 1axj, Z-score 8.2, r.m.s.d. 2.3 Å over 83 Cα atoms), and ferric reductase from Archaeoglobus fulgidus (PDB code 1i0r, Z-score 5.3, r.m.s.d. 2.5 Å for 83 Cα atoms). Comparison of drNimA with a monomer of these structures shows that drNimA contains some unique structural elements, namely the orientation of helix α2 and α3 and the extension of α4, α5, and α6 (compare Fig. 3, a and b, which are in the same orientation). In drNimA, the helices α2 and α3 are involved in the pyruvate binding site, whereas the strand extensions stabilize the homodimer (Fig. 3a). Superposition of these enzymes reveals that the location of the active sites are all on the same side of the barrel, and this region in drNimA contains His-71, which appears to be important for enzymatic activity of the Nim enzymes. Interestingly, the PNPO structure displays a similar dimer
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