Expansion of Substrate Specificity and Catalytic Mechanism of Azoreductase by X-ray Crystallography and Site-directed Mutagenesis
2008; Elsevier BV; Volume: 283; Issue: 20 Linguagem: Inglês
10.1074/jbc.m710070200
ISSN1083-351X
AutoresKosuke Ito, Masayuki Nakanishi, Woo‐Cheol Lee, Yuehua Zhi, Hiroshi Sasaki, Shuhei Zenno, Kaoru Saigo, Yukio Kitade, Masaru Tanokura,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoAzoR is an FMN-dependent NADH-azoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. We previously reported the crystal structure of the enzyme in the oxidized form. In the present study, different structures of AzoR were determined under several conditions to obtain clues to the reaction mechanism of the enzyme. AzoR in its reduced form revealed a twisted butterfly bend of the isoalloxazine ring of the FMN cofactor and a rearrangement of solvent molecules. The crystal structure of oxidized AzoR in a different space group and the structure of the enzyme in complex with the inhibitor dicoumarol were also determined. These structures indicate that the formation of a hydrophobic part around the isoalloxazine ring is important for substrate binding and an electrostatic interaction between Arg-59 and the carboxyl group of the azo compound causes a substrate preference for methyl red over p-methyl red. The substitution of Arg-59 with Ala enhanced the Vmax value for p-methyl red 27-fold with a 3.8-fold increase of the Km value. This result indicates that Arg-59 decides the substrate specificity of AzoR. The Vmax value for the p-methyl red reduction of the R59A mutant is comparable with that for the methyl red reduction of the wild-type enzyme, whereas the activity toward methyl red was retained. These findings indicate the expansion of AzoR substrate specificity by a single amino acid substitution. Furthermore, we built an authentic model of the AzoR-methyl red complex based on the results of the study. AzoR is an FMN-dependent NADH-azoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. We previously reported the crystal structure of the enzyme in the oxidized form. In the present study, different structures of AzoR were determined under several conditions to obtain clues to the reaction mechanism of the enzyme. AzoR in its reduced form revealed a twisted butterfly bend of the isoalloxazine ring of the FMN cofactor and a rearrangement of solvent molecules. The crystal structure of oxidized AzoR in a different space group and the structure of the enzyme in complex with the inhibitor dicoumarol were also determined. These structures indicate that the formation of a hydrophobic part around the isoalloxazine ring is important for substrate binding and an electrostatic interaction between Arg-59 and the carboxyl group of the azo compound causes a substrate preference for methyl red over p-methyl red. The substitution of Arg-59 with Ala enhanced the Vmax value for p-methyl red 27-fold with a 3.8-fold increase of the Km value. This result indicates that Arg-59 decides the substrate specificity of AzoR. The Vmax value for the p-methyl red reduction of the R59A mutant is comparable with that for the methyl red reduction of the wild-type enzyme, whereas the activity toward methyl red was retained. These findings indicate the expansion of AzoR substrate specificity by a single amino acid substitution. Furthermore, we built an authentic model of the AzoR-methyl red complex based on the results of the study. AzoR is an FMN-dependent NADH-azoreductase isolated from Escherichia coli (1Nakanishi M. Yatome C. Ishida N. Kitade Y. J. Biol. Chem. 2001; 276: 46394-46399Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) as a protein responsible for the reduction of azo compounds. AzoR exists as a homodimer composed of 23-kDa subunits. The reaction follows a ping-pong mechanism requiring 2 mol of NADH to reduce 1 mol of methyl red (4′-dimethylaminoazobenzene-2-carboxylic acid), a typical azo dye, into 2-aminobenzoic acid and N,N′-dimethyl-p-phenylenediamine. AzoR also can reduce ethyl red (4′-diethylaminoazobenzene-2-carboxylic acid) and menadione (vitamin K3, 2-methyl-1,4-naphthoquinone) and is inhibited by dicoumarol (3,3′-methylene-bis(4-hydroxycoumarin)). Biochemical studies also revealed that, compared with other azoreductases so far reported, AzoR is different in several regards: in its requirements for cofactors, electron donors, substrate specificity, and amino acid sequence. On the other hand, although the physiological function of AzoR remains unknown, genome projects have inferred its importance from the wide distribution of highly homologous genes in many microorganisms (2Chain P.S. Carniel E. Larimer F.W. Lamerdin J. Stoutland P.O. Regala W.M. Georgescu A.M. Vergez L.M. Land M.L. Motin V.L. Brubaker R.R. Fowler J. Hinnebusch J. Marceau M. Medigue C. Simonet M. Chenal-Francisque V. Souza B. Dacheux D. Elliott J.M. Derbise A. Hauser L.J. Garcia E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13826-13831Crossref PubMed Scopus (481) Google Scholar, 3McClelland M. Sanderson K.E. Clifton S.W. Latreille P. Porwollik S. Sabo A. Meyer R. Bieri T. Ozersky P. McLellan M. Harkins C.R. Wang C. Nguyen C. Berghoff A. Elliott G. Kohlberg S. 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This structure has provided a wealth of information, including the overall fold, the nature of the dimer, and the interactions of the FMN cofactor. A structural similarity search revealed that the overall structure of AzoR resembles that of mammalian NQO1 2The abbreviation used is: NQO1, FAD-dependent NAD(P)H:quinone oxidoreductase 1. 2The abbreviation used is: NQO1, FAD-dependent NAD(P)H:quinone oxidoreductase 1. (FAD-dependent NAD(P)H:quinone oxidoreductase 1, originally called DT-diaphorase) (9Faig M. Bianchet M.A. Talalay P. Chen S. Winski S. Ross D. Amzel L.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3177-3182Crossref PubMed Scopus (170) Google Scholar), ROO from Desulfovibrio gigas (FAD-dependent rubredoxin:oxygen oxidoreductase) (10Frazao C. Silva G. Gomes C.M. Matias P. Coelho R. Sieker L. Macedo S. Liu M.Y. Oliveira S. Teixeira M. Xavier A.V. Rodrigues-Pousada C. Carrondo M.A. Le Gall J. Nat. Struct. 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Because their detailed molecular reaction mechanisms remain largely unknown, AzoR is an interesting case for azoreductase research. Structural and enzymatic insights into AzoR provide knowledge of the molecular mechanism underlying the reduction of azo compounds and would also provide important clues for substrate-specificity expansion techniques that will be essential for industrial use of the enzyme in the biodegradation process of azo compounds. In this study, we have determined the structure of reduced AzoR. This work reveals the structural changes upon reduction of the enzyme and is the first report on the crystal structure of homodimeric flavodoxin-like proteins in the reduced state. In addition, we have determined the crystal structure of oxidized AzoR in a different space group as well as the crystal structure of the enzyme in complex with the inhibitor dicoumarol. Substrate-specificity analysis and site-directed mutagenesis were also performed. According to these analyses, we succeeded in expanding AzoR substrate specificity, and we have built an authentic model of an AzoR-methyl red complex. Finally, the mechanisms underlying the reductive cleavage of azo compounds are discussed. Protein Preparation for Crystallization—The recombinant AzoR used for crystallization was expressed and purified as described previously (1Nakanishi M. Yatome C. Ishida N. Kitade Y. J. Biol. Chem. 2001; 276: 46394-46399Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 21Ito K. Nakanishi M. Lee W.C. Sasaki H. Zenno S. Saigo K. Kitade Y. Tanokura M. Acta Crystallogr. Sect. F Struct. Biol. Crystallalliz. Commun. 2005; 61: 399-402Crossref PubMed Scopus (9) Google Scholar). Improvement of the Quality of the Tetragonal Crystals of Oxidized AzoR—The improved oxidized tetragonal crystals (P42212) were obtained in the same crystallization method as reported previously (21Ito K. Nakanishi M. Lee W.C. Sasaki H. Zenno S. Saigo K. Kitade Y. Tanokura M. Acta Crystallogr. Sect. F Struct. Biol. Crystallalliz. Commun. 2005; 61: 399-402Crossref PubMed Scopus (9) Google Scholar), except that 0.1 mm warfarin (4-hydroxy-3-(3-oxo-1-phenylbutyl)coumarin) was added to the crystallization solution as an additive reagent for crystal growth (the electron density of warfarin was not observed). The crystals grew to full size (0.1 × 0.1 × 0.6 mm) within 1 week. Chemical Reduction of the Tetragonal Crystals of Oxidized AzoR—Crystals of the enzyme in the reduced state were prepared by transferring the oxidized tetragonal crystals to a degassed reducing solution containing 200 mm MgCl2, 100 mm HEPES, pH 7.5, 30% (v/v) 2-propanol, 30% (v/v) ethylene glycol, and a saturated concentration of sodium dithionite at 4 °C. After 10 min, the crystals were transferred to fresh reducing solution. This procedure was repeated two more times. The crystals lost their bright yellow color and became transparent over the course of the treatment. They were then immediately flash-cooled in a stream of nitrogen and stored in liquid nitrogen prior to data collection. It was verified that the crystal remained transparent after data collection. Co-crystallization with Dicoumarol—The purified protein was dialyzed against a solution containing 10 mm Tris-HCl, pH 8.0, 0.1 mm FMN, 4% (v/v) pyridine, and 2 mm dicoumarol. Crystals of AzoR in complex with dicoumarol were obtained from a drop made by mixing 16 mg/ml of the protein solution mentioned above and an equal volume of reservoir solution containing 200 mm NaCl, 100 mm HEPES, pH 7.5, and 20% (w/v) polyethylene glycol 3000. The drop was equilibrated over the reservoir solution by the hanging-drop vapor diffusion method at 20 °C. The crystals grew to full size (0.05 × 0.5 × 0.5 mm) within 1 week. These crystals belonged to the tetragonal space group P4212. Crystallization of the Orthorhombic Crystals of Oxidized AzoR—The purified protein was dialyzed against a solution containing 10 mm Tris-HCl, pH 8.0, and 0.1 mm FMN. The orthorhombic (P212121) crystals of oxidized AzoR were obtained from a drop made by equal volumes of three solutions: 8 mg/ml of the protein solution mentioned above, 100 mm NAD+ solution (the electron density of NAD+ was not observed), and a reservoir solution containing 200 mm NaOAc, 200 mm sodium cacodylate, pH 6.7, 15% (w/v) polyethylene glycol 8000, and 3% (v/v) dimethyl sulfoxide. The drop was equilibrated over the reservoir solution by the hanging-drop vapor diffusion method at 25 °C. The crystals grew to full size (0.03 × 0.05 × 0.5 mm) within 2 weeks. Data Collection and Processing—All diffraction data were collected under cryogenic conditions at 100 K. Prior to data collection, the crystals were soaked in a reservoir solution containing 30% (v/v) ethylene glycol or 25% (v/v) glycerol as a cryo-protectant. All diffraction data were collected at KEK (Tsukuba, Japan). The beamlines used are shown in Table 1. Data were reduced with MOSFLM, SCALA, and TRUNCATE from the CCP4 program suite (22Collaborative Computational Project Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19668) Google Scholar).TABLE 1Data collection and refinement statistics for AzoR The values in parentheses are for the highest resolution shell. R.m.s., root mean square.Oxidized AzoRReduced AzoRAzoR-dicoumarolData collection Space groupP42212P212121P42212P4212 Unit cell (Å)a = b = 92.20, c = 51.74a = 55.07, b = 89.89, c = 101.87a = b = 91.88, c = 51.62a = b = 95.50, c = 54.50 Resolution range (Å)34.30–1.40 (1.48–1.40)41.17–2.10 (2.21–2.10)29.06–1.70 (1.79–1.70)19.89–2.30 (2.42–2.30) Unique reflections4230729939240039493 Redundancy8.9 (6.9)4.0 (3.9)7.5 (5.9)16.3 (16.0) Completeness (%)96.3 (94.2)99.3 (99.9)96.8 (81.1)81.8 (85.0) Average I/σ(I)9.0 (2.6)8.7 (2.7)8.1 (2.6)6.5 (2.8) RmergeaRmerge = ΣhklΣi |Ii(hkl) – 〈I(hkl)〉|/ΣhklΣi Ii(hkl), where Ii(hkl) is the i-th intensity measurement of reflection hkl, including symmetry-related reflections, and 〈I(hkl)〉 is its average (%)4.7 (27.4)7.5 (27.9)6.0 (27.6)9.4 (27.2) BeamlinePF BL18BNW12 PF-ARPF BL6APF BL6ARefinement Rbr = 100 Σ||Fobs| – |Fcalc||/ Σ|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, respectively/RfreecRfree was calculated by using 5% of randomly selected reflections that were excluded from the refinement (%)18.4/22.017.0/21.418.5/21.617.6/22.0 R.m.s. bond length (Å)0.0460.0170.0170.012 R.m.s. bond angle (°)3.7391.4381.6131.205 Copies in asymmetric unit1211 No. of molecules/Average B factor (Å2) Amino acid residues1–58, 65–200/17.21–200, 1–200/29.61–57, 65–200/19.11–59, 63–200/38.9 Solvent molecules119/24.9164/31.3111/26.669/37.4 FMN (dicoumarol)1/10.6 (–)2/22.4 (–)1/12.6 (–)1/30.5 (1/43.1)a Rmerge = ΣhklΣi |Ii(hkl) – 〈I(hkl)〉|/ΣhklΣi Ii(hkl), where Ii(hkl) is the i-th intensity measurement of reflection hkl, including symmetry-related reflections, and 〈I(hkl)〉 is its averageb r = 100 Σ||Fobs| – |Fcalc||/ Σ|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, respectivelyc Rfree was calculated by using 5% of randomly selected reflections that were excluded from the refinement Open table in a new tab Structure Determination—The initial structures of all the crystals were obtained by molecular replacement with MOLREP (23Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4098) Google Scholar) using the 1.8 Å resolution structure of oxidized AzoR as a search model (8Ito K. Nakanishi M. Lee W.C. Sasaki H. Zenno S. Saigo K. Kitade Y. Tanokura M. J. Biol. Chem. 2006; 281: 20567-20576Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The solutions were then improved by ARP/wARP (24Perrakis A. Morris R. Lamzin V.S. Nature Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2561) Google Scholar) followed by iterative manual model building with XtalView (25McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2016) Google Scholar). All the stages of maximum likelihood refinement were carried out with REFMAC5 (26Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13712) Google Scholar). For the refinement of the oxidized and reduced tetragonal crystal structures, the restraint of the planarity of the isoalloxazine ring was removed from the standard REFMAC5 library to allow the model to adopt the omit density map more precisely. Model Analysis—The quality of the model was checked with PROCHECK (27Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. App. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). LSQKAB was used to superpose the molecules and to calculate the root mean square deviation between pairs of equivalent Cα atoms and all atoms of the proteins (28Kabsch W. Acta Crystallogr. Sect. A. 1976; 32: 922-923Crossref Scopus (2274) Google Scholar). Structure figures were prepared with PyMOL (29DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar). Mutant Preparations—For enzymatic analyses, mutations and C-terminal His tag were introduced into the AzoR gene by two rounds of PCR with pETacpD as a template (1Nakanishi M. Yatome C. Ishida N. Kitade Y. J. Biol. Chem. 2001; 276: 46394-46399Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The NADH-methyl red reductase activity of the His-tagged wild-type AzoR was very similar to that of the non-tagged wild-type AzoR. To mutate Arg-59 to Ala, the following pairs of oligonucleotide primers were used for the first PCR: 5′-terminal sense primer for AzoR (5′-GGGAATTCCATATGAGCAAGGTATTAGTTCTTAAATCCAGC-3′ containing an NdeI site) and R59A mutation antisense primer (5′-CGGCGCATCGCTCGGAGCCAGAGCGCCAACCAGTTC-3′) were used to amplify the 5′-terminal part of the AzoR gene. R59A mutation sense primer (5′-GAACTGGTTGGCGCTCTGGCTCCGAGCGATGCGCCG-3′) and 3′-terminal antisense primer for AzoR (5′-AAACCGCTCGAGTTAGTGATGGTGATGGTGGTGTGCAGAAACAATGCTGTCGATGGC-3′ containing an XhoI site and His6 tag sequence) were used to amplify the 3′-terminal part of the AzoR gene. PCR products were used as templates for the second PCR, which used the 5′-terminal sense primer and 3′-terminal antisense primer for AzoR. To generate other mutants, the following primers were used in place of R59A mutation sense and antisense primers: Y120A mutation sense primer (5′-GCAGGCGTTACTTTCCGCGCTACCGAGAACGGTCCG-3′) and Y120A mutation antisense primer (5′-CGGACCGTTCTCGGTAGCGCGGAAAGTAACGCCTGC-3′) for Tyr-120 to Ala mutant; F162A mutation sense primer (5′-CCACGTTCCTCGGCGCTATCGGCATTACCGATG-3′) and F162A mutation antisense primer (5′-CATCGGTAATGCCGATAGCGCCGAGGAACGTGG-3′) for Phe-162 to Ala mutant. To generate the His-tagged wild-type AzoR gene as a control that is catalytically very similar to AzoR, the 5′-terminal sense primer and the 3′-terminal antisense primer for AzoR described above were used for PCR with pETacpD as a template. The PCR product of each mutated AzoR was inserted between the NdeI and the XhoI sites of the expression vector pET-22b (Novagen). The entire DNA sequence was confirmed by DNA sequencing. His-tagged wild-type and mutant AzoR proteins were expressed in E. coli BL21(DE3) at 37 °C in Luria Bertani medium containing 100 μg/ml ampicillin. Protein expression was induced by adding 1 mm isopropyl-β-d-thiogalactopyranoside to early exponential phase cultures (A600 ∼ 0.5) for 3 h. Bacteria were lysed in a solution containing 50 mm Tris-HCl, pH 7.5, 2 mm 2-mercaptoethanol, 500 mm NaCl, 20 mm imidazole, and 10 mg/ml lysozyme by sonication. The lysate was centrifuged, and the His-tagged proteins were purified by gravity-flow chromatography using nickel-nitrilotriacetic acid-agarose (Qiagen) according to the manufacturer's instructions. Eluted proteins were stored at -80 °C in a solution containing 20 mm Tris-HCl, pH 7.5, 2 mm 2-mercaptoethanol, 200 mm NaCl, and 0.1 mm FMN. Protein samples were analyzed on SDS-PAGE and were more than 95% pure. Enzyme Assays—The NADH-methyl red reductase activity for each mutant was determined spectrophotometrically by a method described previously (1Nakanishi M. Yatome C. Ishida N. Kitade Y. J. Biol. Chem. 2001; 276: 46394-46399Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The initial reaction rates were fitted to the Equation 11v=1V+KAVA+KBVB([Eq. 1]) where v is the initial reaction rate, V is the maximum reaction rate at infinite substrate concentrations, A and B are the concentrations of methyl red and NADH, respectively, and KA and KB are their corresponding Michaelis constants. The initial reaction rate of p-methyl red reduction was determined in the same manner as methyl red, except that a 460-nm wavelength was used to monitor the decrease in absorbance of p-methyl red and a molar absorption coefficient of 17310 m-1 cm-1 was used. KB was applied to obtain Michaelis constants for p-methyl red because KB does not depend on the kind of azo compound. All of these assays were performed in triplicate. Structures of Oxidized and Reduced AzoR in Tetragonal Crystals—The structure of oxidized AzoR in tetragonal crystal was previously determined at 1.8 Å resolution (8Ito K. Nakanishi M. Lee W.C. Sasaki H. Zenno S. Saigo K. Kitade Y. Tanokura M. J. Biol. Chem. 2006; 281: 20567-20576Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). We have now improved the crystallization conditions and increased the resolution to 1.4 Å. In addition, we have determined the structure of reduced AzoR at 1.7 Å resolution using chemically reduced tetragonal crystals. The results allow the stringent comparison of the two states, especially the conformation of the isoalloxazine ring of FMN. The structure of this ring was unambiguously determined without the restraint of planarity for both the oxidized and reduced enzymes (Fig. 1). The reduced crystals turned colorless, indicating that two-electron reduction of the flavin occurred. The data collection and the final refinement statistics are summarized in Table 1. The AzoR structures are nearly identical between the oxidized and reduced states. The root mean square deviations between equivalent 193 Cα atoms and all atoms of the amino acid residues, except hydrogens, in the two states are 0.282 and 0.582 Å, respectively. However, prominent structural differences are found in the active site. In the oxidized enzyme, the isoalloxazine ring of the flavin is nearly planar but shows a slight twist conformation (Fig. 1, A and C). Upon reduction of the enzyme, the isoalloxazine ring adopts a butterfly bend conformation along the N5-N10 axis caused by a shift of the dimethyl and pyrimidine rings toward the re-face by an angle of ∼15° (Fig. 1, B, D, and F). The slight twist conformation found in the oxidized state is retained in the reduced state, and N5 and N10 of the isoalloxazine ring move up toward the si-side. Therefore, the central ring of the isoalloxazine is distorted to a twist-boat conformation upon reduction. The movement of N10 is small relative to that of N5. The other structural changes associated with reduction are a significant movement of a water molecule and a loss of two spherical electron density peaks on FMN. The water molecule, which is hydrogen-bonded to Oδ-1 of Asn-97 in the oxidized state, still forms a hydrogen bond in the reduced state (Fig. 1E). This water molecule, however, moves by 2.4 Å and makes an additional hydrogen bond with N5 of the isoalloxazine ring upon reduction. The protonation of the N5 of the reduced isoalloxazine ring has been proved biochemically (30Muller F. Muller F Chemistry and Biochemistry of Flavoenzymes. 1. CRC Press, Inc., Boca Raton, FL1991: 1-71Google Scholar). This hydrogen bond may help stabilize the upward movement of the N5 atom, which adopts sp 3AzoR is a homodimeric enzyme (1Nakanishi M. Yatome C. Ishida N. Kitade Y. J. Biol. Chem. 2001; 276: 46394-46399Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The redox center FMN is found in the dimer interface, and both monomers contribute to form the two identical catalytic sites (8Ito K. Nakanishi M. Lee W.C. Sasaki H. Zenno S. Saigo K. Kitade Y. Tanokura M. J. Biol. Chem. 2006; 281: 20567-20576Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). When we refer to one catalytic site, residues will be primed (one monomer) or non-primed (the other monomer), but they areequivalent. NQO1 is also a homodimeric enzyme in a similar fashion to AzoR (36Li R. Bianchet M.A. Talalay P. Amzel L.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8846-8850Crossref PubMed Scopus (307) Google Scholar). Thus, primes will be used in a similar way as with AzoR. hybridization in the reduced state (31Beinert W.D. Ruterjans H. Muller F. Eur. J. Biochem. 1985; 152: 573-579Crossref PubMed Scopus (42) Google Scholar). The two spherical electron density peaks on FMN, which were also found in the oxidized 1.8 Å resolution structure as reported previously, are currently modeled by water molecules, although they are slightly larger than the electron density peaks of water molecules. The loss of two peaks upon reduction may be attributable to electronic restructuring of the reduced flavin. The Conformational Change of the Isoalloxazine Ring and the Solvent Rearrangement upon Reduction—The butterfly bending direction in reduced AzoR is the opposite of the conformation predicted for reduced free flavin (32Dixon D.A. Lindner D.L. Branchaud B. Lipscomb W.N. Biochemistry. 1979; 18: 5770-5775Crossref PubMed Scopus (58) Google Scholar, 33Zheng Y.J. Ornstein R.L. J. Am. Chem. Soc. 1996; 118: 9402-9408Crossref Scopus (119) Google Scholar, 34Nakai S. Yoneda F. Yamabe T. Theor. Chem. Acc. 1999; 103: 109-116Crossref Scopus (16) Google Scholar). This considerable disagreement suggests that the peptide moiety may greatly influence the equilibrium conformation of the isoalloxazine system in AzoR. On the other hand, although it is not clear why the central ring of the isoalloxazine adopts the twist-boat form in the reduced enzyme, which is energetically less favorable than a chair form, this conformation would affect the redox potential of AzoR. In addition, because free oxidized isoalloxazine is planar (35Trus B.L. Fritchie C.J. Acta Crystallogr. Sect. B Struct. Sci. 1969; 25: 1911-1918Crossref PubMed Scopus (14) Google Scholar), the twisted form of the oxidized isoalloxazine of AzoR may favor reduction. In the reduced state of the enzyme, the up-moved protonated N5 of the isoalloxazine ring is stabilized by the hydrogen bond with Oδ-1 of Asn-97 through a bridging water molecule (Fig. 1E). The protonation of the N5 of the reduced isoalloxazine ring has been proved biochemically (30Muller F. Muller F Chemistry and Biochemistry of Flavoenzymes. 1. CRC Press, Inc., Boca Raton, FL1991: 1-71Google Scholar). If methyl red binds to reduced AzoR on top of the isoalloxazine ring as described below, this water molecule must be replaced, resulting in the disruption of the hydrogen bond network. This may cause destabilization of the protonated state of the N5 atom, and a hydride anion would then be released efficiently from the N5 position to an electron acceptor azo compound. Structure of AzoR in Complex with Dicoumarol—Dicoumarol inhibits the azo reduction activity of AzoR (1Nakanishi M. Yatome C. Ishida N. Kitade Y. J. Biol. Chem. 2001; 276: 46394-46399Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). To obtain clues to the substrate binding mode, we co-crystallized AzoR with dicoumarol and have determined the structure at 2.3 Å resolution. Although the average completeness of the data of the AzoR-dicoumarol complex is 81.1% (Table 1), there was not any uninterpretable electron density region in model building. The crystal structure shows that dicoumarol is bound to the space above the isoalloxazine ring mainly by hydrophobic interactions (Fig. 2A). In their interactions, the two coumarin rings of dicoumarol are sandwiched as a result of ring-stacking interactions. The one ring is sandwiched between the phenyl group of Phe-162′ and the isoa
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