Three-dimensional Structure of AzoR from Escherichia coli

The crystal structure of AzoR (azoreductase) has been determined in complex with FMN for two different crystal forms at 1.8 and 2.2Å resolution. AzoR is an oxidoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. This enzyme is an FMN-dependent NADH-azoreductase and catalyzes the reductive cleavage of azo groups by a ping-pong mechanism. The structure suggests that AzoR acts in a homodimeric state forming the two identical catalytic sites to which both monomers contribute. The structure revealed that each monomer of AzoR has a flavodoxin-like structure, without the explicit overall amino acid sequence homology. Superposition of the structures from the two different crystal forms revealed the conformational change and suggested a mechanism for accommodating substrates of different size. Furthermore, comparison of the active site structure with that of NQO1 complexed with substrates provides clues to the possible substrate-binding mechanism of AzoR. The crystal structure of AzoR (azoreductase) has been determined in complex with FMN for two different crystal forms at 1.8 and 2.2Å resolution. AzoR is an oxidoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. This enzyme is an FMN-dependent NADH-azoreductase and catalyzes the reductive cleavage of azo groups by a ping-pong mechanism. The structure suggests that AzoR acts in a homodimeric state forming the two identical catalytic sites to which both monomers contribute. The structure revealed that each monomer of AzoR has a flavodoxin-like structure, without the explicit overall amino acid sequence homology. Superposition of the structures from the two different crystal forms revealed the conformational change and suggested a mechanism for accommodating substrates of different size. Furthermore, comparison of the active site structure with that of NQO1 complexed with substrates provides clues to the possible substrate-binding mechanism of AzoR. Azo dyes are widely used colorants for printing, textile dyeing, food preparation, cosmetic production, and clinical purposes because of their chemical stability, ease of synthesis, and utility (1.Meyer U. FEMS Symp. 1981; 12: 371-385Google Scholar). However, there is considerable concern about the toxicity, and especially the carcinogenicity, of some azo dyes (2.Holme I. Griffiths J. Ecological Aspects of Color Chemistry: Developments in the Chemistry and Technology of Organic Dyes. Society of Chemistry Industry, Oxford1984: 111-128Google Scholar). These compounds are frequently found in a chemically unchanged form even after wastewater treatment (3.Levine W.G. Drug Metab. Rev. 1991; 23: 253-309Crossref PubMed Scopus (216) Google Scholar), resulting in environmental pollutants. Therefore, efficient degradation systems for azo dyes should be established. Degradation systems based on chemical procedures are expensive, require much energy, and often yield hazardous byproducts. On the other hand, biological degradation using microorganisms can decompose azo dyes under mild conditions, without the problems described above (4.Robinson T. McMullan G. Marchant R. Nigam P. Bioresour. Technol. 2001; 77: 247-255Crossref PubMed Scopus (4291) Google Scholar, 5.Stolz A. Appl. Microbiol. Biotechnol. 2001; 56: 69-80Crossref PubMed Scopus (946) Google Scholar). To facilitate the development of biodegradation systems, it is essential to understand the detailed mechanisms of dye-degrading enzymes. AzoR is an oxidoreductase isolated from Escherichia coli (6.Nakanishi M. Yatome C. Ishida N. Kitade Y. J. Biol. Chem. 2001; 276: 46394-46399Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) as a protein responsible for the reduction of azo compounds. Biochemical studies have revealed that AzoR catalyzes the reductive cleavage of azo groups (-N=N-) utilizing NADH but not NADPH as an electron donor by means of the flavin cofactor FMN. 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. Therefore, it is thought that two cycles of the ping-pong mechanism were required for the reductive cleavage of azo groups. However, details about the molecular mechanism of the catalysis remain unknown. Many kinds of bacterial azoreductases have been isolated and characterized for the progress of bioremediation (7.Zimmermann T. Kulla H.G. Leisinger T. Eur. J. Biochem. 1982; 129: 197-203Crossref PubMed Scopus (376) Google Scholar, 8.Zimmermann T. Gasser F. Kulla H.G. Leisinger T. Arch. Microbiol. 1984; 138: 37-43Crossref PubMed Scopus (118) Google Scholar, 9.Ghosh D.K. Mandal A. Chaudhuri J. FEMS Microbiol. Lett. 1992; 77: 229-233Crossref PubMed Scopus (52) Google Scholar, 10.Ghosh D.K. Ghosh S. Sadhukhan P. Mandal A. Chaudhuri J. Indian J. Exp. Biol. 1993; 31: 951-954PubMed Google Scholar, 11.Rafii F. Cerniglia C.E. Appl. Environ. Microbiol. 1993; 59: 1731-1734Crossref PubMed Google Scholar, 12.Suzuki Y. Yoda T. Ruhul A. Sugiura W. J. Biol. Chem. 2001; 276: 9059-9065Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 13.Moutaouakkil A. Zeroual Y. Zohra Dzayri F. Talbi M. Lee K. Blaghen M. Arch. Biochem. Biophys. 2003; 413: 139-146Crossref PubMed Scopus (105) Google Scholar, 14.Maier J. Kandelbauer A. Erlacher A. Cavaco-Paulo A. Gubitz G.M. Appl. Environ. Microbiol. 2004; 70: 837-844Crossref PubMed Scopus (209) Google Scholar, 15.Ramalho P.A. Paiva S. Cavaco-Paulo A. Casal M. Cardoso M.H. Ramalho M.T. Appl. Environ. Microbiol. 2005; 71: 3882-3888Crossref PubMed Scopus (37) Google Scholar). However, AzoR is different from other azoreductases reported thus far with respect to its requirements for cofactors, electron donors, and substrate specificity and its amino acid sequence. In addition, although the physiological function of AzoR remains unknown, its importance has been deduced from genome projects that have revealed the wide distribution of highly homologous genes in many microorganisms. For example, these genes are found in Yersinia pseudotuberculosis (16.Chain 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 (489) Google Scholar), Salmonella enterica (17.McClelland 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. Strong C. Du F. Carter J. Kremizki C. Layman D. Leonard S. Sun H. Fulton L. Nash W. Miner T. Minx P. Delehaunty K. Fronick C. Magrini V. Nhan M. Warren W. Florea L. Spieth J. Wilson R.K. Nat. Genet. 2004; 36: 1268-1274Crossref PubMed Scopus (307) Google Scholar), Photorhabdus luminescens (18.Duchaud E. Rusniok C. Frangeul L. Buchrieser C. Givaudan A. Taourit S. Bocs S. Boursaux-Eude C. Chandler M. Charles J.F. Dassa E. Derose R. Derzelle S. Freyssinet G. Gaudriault S. Medigue C. Lanois A. Powell K. Siguier P. Vincent R. Wingate V. Zouine M. Glaser P. Boemare N. Danchin A. Kunst F. Nat. Biotechnol. 2003; 21: 1307-1313Crossref PubMed Scopus (483) Google Scholar), Vibrio vulnificus (19.Chen C.Y. Wu K.M. Chang Y.C. Chang C.H. Tsai H.C. Liao T.L. Liu Y.M. Chen H.J. Shen A.B. Li J.C. Su T.L. Shao C.P. Lee C.T. Hor L.I. Tsai S.F. Genome Res. 2003; 13: 2577-2587Crossref PubMed Scopus (307) Google Scholar), Haemophilus influenzae (20.Harrison A. Dyer D.W. Gillaspy A. Ray W.C. Mungur R. Carson M.B. Zhong H. Gipson J. Gipson M. Johnson L.S. Lewis L. Bakaletz L.O. Munson Jr., R.S. J. Bacteriol. 2005; 187: 4627-4636Crossref PubMed Scopus (169) Google Scholar), Pseudomonas putida (21.Nelson K. Paulsen I. Weinel C. Dodson R. Hilbert H. Fouts D. Gill S. Pop M. Martins Dos Santos V. Holmes M. Brinkac L. Beanan M. DeBoy R. Daugherty S. Kolonay J. Madupu R. Nelson W. White O. Peterson J. Khouri H. Hance I. Lee P. Holtzapple E. Scanlan D. Tran K. Moazzez A. Utterback T. Rizzo M. Lee K. Kosack D. Moestl D. Wedler H. Lauber J. Hoheisel J. Straetz M. Heim S. Kiewitz C. Eisen J. Timmis K. Duesterhoft A. Tummler B. Fraser C. Environ. Microbiol. 2002; 4: 799-808Crossref PubMed Scopus (1052) Google Scholar), and so forth. On the other hand, AzoR has the ability to reduce menadione (vitamin K3, 2-methyl-1,4-naphthoquinone) (6.Nakanishi M. Yatome C. Ishida N. Kitade Y. J. Biol. Chem. 2001; 276: 46394-46399Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Quinone compounds are biologically active molecules that function as lipid electron carriers. Especially, a derivative of vitamin K is predominantly employed during anaerobic respiration in E. coli (22.Unden G. Bongaerts J. Biochim. Biophys. Acta. 1997; 1320: 217-234Crossref PubMed Scopus (536) Google Scholar). These facts imply that AzoR plays an essential role in electron transport or metabolism during redox reaction or other processes. AzoR is representative of a poorly characterized family of azo dye reductases. The structural determination of AzoR would be a first step toward elucidating its molecular mechanism of function, as well as those of highly homologous proteins of AzoR in other species, and the development of the biodegradation system. Here we report the crystal structures of AzoR complexed with FMN in two different crystal forms at 1.8 and 2.2 Å resolution. The structures revealed that AzoR has a flavodoxin-like structure without the explicit overall amino acid sequence homology and would likely act as a homodimeric FMN-containing enzyme. Superposition of the two crystal structures revealed the regions that participate in the conformational change of the active site, which would be a mechanism for accommodating substrates of different sizes. Furthermore, structural comparison of the active site with that of NQO1 revealed the conservation of amino acid residues, suggesting the substrate binding mode of AzoR. This is the first report of the structure of AzoR orthologues and of FMN-dependent NADH-azoreductase. Protein Preparation and Crystallization—The recombinant E. coli AzoR used in this paper was expressed and purified as described previously (6.Nakanishi M. Yatome C. Ishida N. Kitade Y. J. Biol. Chem. 2001; 276: 46394-46399Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Two crystal forms, P42212 and P4212, of the AzoR were obtained under different crystallization conditions that contained FMN. The P42212 crystals were obtained according to the published method (23.Ito K. Nakanishi M. Lee W.C. Sasaki H. Zenno S. Saigo K. Kitade Y. Tanokura M. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2005; 61: 399-402Crossref PubMed Scopus (9) Google Scholar). The P4212 crystals were obtained from a drop made by mixing a solution containing 23 mg/ml protein in 10 mm Tris-HCl (pH 8.0), 1 mm FMN, and an equal volume of reservoir solution containing 200 mm NaCl, 100 mm CAPS 2The abbreviations used are: CAPS, N-cyclohexyl-3-aminopropanesulfonic acid; r.m.s., root mean square; SIRAS, single isomorphous replacement with anomalous scattering; NQO1, NAD(P)H:quinone oxidoreductase 1; ROO, rubredoxin:oxygen oxidoreductase. (pH 10.5), 20% (w/v) polyethylene glycol 8000, 20% (v/v) 1,4-dioxane, and 4 mm menadione. The drop was equilibrated over 500 μl of the reservoir solution by the hanging drop vapor diffusion method at 293 K. The crystals grew to full size (0.08 × 0.6 × 0.6 mm) within 2 weeks. Preparation of Heavy Atom Derivative—To prepare the heavy atom derivative, the P42212 crystals were soaked for 23 days in a solution prepared by diluting 1 part K2PtCl4-saturated reservoir solution with 10 parts reservoir solution at 288 K. The details of the screening of heavy atom derivatives were described in a previous report (23.Ito K. Nakanishi M. Lee W.C. Sasaki H. Zenno S. Saigo K. Kitade Y. Tanokura M. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2005; 61: 399-402Crossref PubMed Scopus (9) Google Scholar). Data Collection and Processing—Prior to data collection, P42212 crystals and the K2PtCl4 derivative were soaked in a reservoir solution containing 30% (v/v) ethylene glycol as a cryoprotectant. The P4212 crystals were soaked in a reservoir solution containing 25% (v/v) glycerol as a cryoprotectant. The native data of the P42212 and P4212 crystals were measured at BL6A of the Photon Factory, KEK (Tsukuba, Japan) using an ADSC Quantum 4 CCD detector, and the K2PtCl4 derivative data were collected at the BL41XU of SPring-8 (Harima, Japan) using a Mar Research 165-mm CCD detector. All of the diffraction data were collected under cryogenic conditions at 100 K. The data were reduced with MOSFLM, SCALA, and TRUNCATE from the CCP4 program suite (24.Collaborative Computational Project, Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar). Structure Solution and Phasing—The initial structure in the P42212 crystal was determined by the SIRAS method using the K2PtCl4 derivative. The heavy atom parameters and phases were calculated with SOLVE (25.Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). Subsequently, maximum likelihood density modification was performed with RESOLVE (26.Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 965-972Crossref PubMed Scopus (1634) Google Scholar), and the phases were further improved and extended to a resolution of 65.2-1.8 Å with ARP/wARP (27.Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2564) Google Scholar). The initial structure in the P4212 crystal was determined by molecular replacement. The calculation of the molecular replacement was carried out with MOLREP (28.Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4153) Google Scholar) using the structure of the P42212 crystal as a search model. The high resolution limit of the diffraction data were set to 3.0 Å, and the low resolution limit was selected according to the standard protocol of MOLREP. The solution was then improved by rigid body refinement with REFMAC5 (29.Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13870) Google Scholar). The phases were improved and extended to a resolution of 67.4-2.2 Å with ARP/wARP. Model Building and Refinement—ARP/wARP was used for automatic model building followed by iterative manual model building with XtalView (30.McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2022) Google Scholar). 2mFo - DFc and mFo - DFc SIGMAA-weighted electron density maps (31.Read R.J. Acta Crystallogr. Sect. A. 1986; 42: 140-149Crossref Scopus (2036) Google Scholar) were used as references. All stages of maximum likelihood refinement were carried out with REFMAC5. The P42212 crystal structure consisted of 197 residues (1-59 and 63-200) of a possible 200 residues, one FMN, one ethylene glycol, one 2-propanol, and a total of 140 water molecules in an asymmetric unit. Arg-58 was modulated to Ala, and loop residues 60-62 were not built because of their poor electron density. The structure from the P4212 crystal included all possible amino acid residues, one FMN, two glycerols, and a total of 66 water molecules in an asymmetric unit. Although menadione was included in the crystallization solution for the P4212 crystals, its electron density was not observed. Model Analysis—The quality of the model was checked using PROCHECK (32.Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The superposition of molecules and calculation of the r.m.s. deviation between pairs of equivalent Cα atoms and all atoms of proteins were executed using LSQKAB (33.Kabsch W. Acta Crystallogr. Sect. A. 1976; 32: 922-923Crossref Scopus (2358) Google Scholar). Secondary structures were assigned using DSSP (34.Kabsch W. Sander C. Biopolymers. 1983; 12: 2577-2637Crossref Scopus (12333) Google Scholar). Figure Preparation—The figures were prepared using PyMOL (35.DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar), LIGPLOT (36.Wallace A.C. Laskowski R.A. Thornton J.M. Protein Eng. 1995; 8: 127-134Crossref PubMed Scopus (4368) Google Scholar), and GRASP (37.Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-296Crossref PubMed Scopus (5316) Google Scholar). Overall Structure—Both forms of the AzoR crystal contain one 23-kDa monomer in an asymmetric unit, and the structures in both crystal forms are similar except with respect to a few regions (see below). Each monomer in both crystal forms constitutes a homodimer by a crystallographic symmetry operation in the same manner. The redox center FMN is found at the dimer interface, and both monomers contribute to form the two identical catalytic sites by the distance in ∼25 Å away. These facts are consistent with the previous report that AzoR exists as a homodimer in solution, as shown by analytical gel filtration (6.Nakanishi M. Yatome C. Ishida N. Kitade Y. J. Biol. Chem. 2001; 276: 46394-46399Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Throughout this report, positions in the two monomers are distinguished by primed and nonprimed characters. The structure from the P4212 crystal includes all possible amino acid residues, giving the overall structure shown in Fig. 1 with the secondary structure assignment. The structure revealed that each monomer of AzoR has a flavodoxin-like structure, although a marginal level of sequence homology has been found only in the active site region. AzoR has a structure in which five parallel β-strands (β1, β2, β3, β8, and β9) form an open twisted central β-sheet surrounded on both sides by a total of six helices (α1-α6). Helices α1 and α6 are on one side of the β-sheet, and helices α2, α3, α4, and α5 are located on the opposite side. The dimerization occurs mainly via anti-parallel side-to-side packing of the loop L7-helix α4 region and loop L11-helix α5 region of each monomer. The loop L3-helix α2 region also participates in the dimerization and interacts with the loop L3′-helix α2′ region of the other monomer. A summary of the data collection and SIRAS phasing statistics is presented in Table 1. The final refinement statistics are summarized in Table 2.TABLE 1Statistics for data collection and heavy atom phasingNative (P42212)Native (P4212)K2PtCl4 derivativeData collectionBeamlinePF-BL6APF-BL6ASPring-8 BL41XUWavelength (Å)1.0001.0001.000Space groupP42212P4212P42212Unit cell parameters (Å)a = b = 92.19, c = 51.85a = b = 94.63, c = 54.12a = b = 92.38, c = 51.67Resolution range (Å)64.55-1.80 (1.90-1.80)67.42-2.20 (2.32-2.20)65.94-2.50 (2.64-2.50)No. of observations28781163326106879No. of unique observations21239121458145Completeness (%)99.7 (98.2)93.8 (83.8)99.7 (100.0)Redundancy13.6 (8.3)5.2 (4.9)13.1 (13.5)Average I/σ(I)7.0 (3.0)6.4 (2.6)11.4 (8.6)Rmerge (%)aRmerge of the native data is as follows: Rmerge = ∑hkl∑i|Ii(hkl) - 〈I(hkl) 〉|/∑hkl∑i Ii(hkl), where Ii(hkl) is the ith intensity measurement of reflection hkl, including symmetry-related reflections, and 〈I(hkl) 〉 is its average. Rmerge of the K2PtCl4 derivative data is defined in the same way as Rmerge of the native data, except that the summation is executed individually for each Bijvoet pair.0.063 (0.225)0.090 (0.284)0.047 (0.078)Ranom (%)bRanom = ∑hkl| 〈I+(hkl) 〉 - 〈I−(hkl) 〉|/| 〈I+(hkl) 〉 + 〈I−(hkl) 〉|, where 〈I+(hkl) 〉 and 〈I−(hkl) 〉 are the averages of I+(hkl) and I−(hkl) respectively.0.021 (0.025)Heavy atom phasingResolution20.0-2.5 ÅScaling R factor (%)cScaling R factor (Riso) = ∑hkl||Fderiv(hkl)| - |Fnative(hkl)||/∑hkl|Fnative(hkl)|.22.6Number of heavy atom sites1FOM (SOLVE)dThe figure of merit (FOM) as defined in SOLVE and RESOLVE.0.30FOM (RESOLVE)dThe figure of merit (FOM) as defined in SOLVE and RESOLVE.0.60a Rmerge of the native data is as follows: Rmerge = ∑hkl∑i|Ii(hkl) - 〈I(hkl) 〉|/∑hkl∑i Ii(hkl), where Ii(hkl) is the ith intensity measurement of reflection hkl, including symmetry-related reflections, and 〈I(hkl) 〉 is its average. Rmerge of the K2PtCl4 derivative data is defined in the same way as Rmerge of the native data, except that the summation is executed individually for each Bijvoet pair.b Ranom = ∑hkl| 〈I+(hkl) 〉 - 〈I−(hkl) 〉|/| 〈I+(hkl) 〉 + 〈I−(hkl) 〉|, where 〈I+(hkl) 〉 and 〈I−(hkl) 〉 are the averages of I+(hkl) and I−(hkl) respectively.c Scaling R factor (Riso) = ∑hkl||Fderiv(hkl)| - |Fnative(hkl)||/∑hkl|Fnative(hkl)|.d The figure of merit (FOM) as defined in SOLVE and RESOLVE. Open table in a new tab TABLE 2Refinement statisticsP42212P4212Resolution range (Å)64.55-1.8067.42-2.20Reflections20,09511,523R/Rfree (%)aR = 100 ∑||Fobs - Fcac||/∑|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, respectively. Rfree was calculated by using 5% of randomly selected reflections that were excluded from the refinement.19.4/23.416.9/20.5Number of MoleculesAmino acid residues197200FMN11Ethylene glycol12-Propanol1Glycerol2Water14066Average B factor (Å2)Amino acid residues21.031.4FMN13.218.6Ethylene glycol29.12-Propanol28.6Glycerol32.5Water21.528.6r.m.s. deviation from ideal valuesBond length (Å)0.0240.031Bond angle (°)1.9632.200Ramachandran plot (%)In most favored regions91.890.8In allowed regions7.68.6In generously allowed regions0.60.6In disallowed regions0.00.0a R = 100 ∑||Fobs - Fcac||/∑|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, respectively. Rfree was calculated by using 5% of randomly selected reflections that were excluded from the refinement. Open table in a new tab FMN Binding Site—The FMN prosthetic groups bind on the C-terminal end of the central β-sheet at the dimer interface. Each FMN cofactor binds to both monomers; 15 hydrogen bonds are formed with one monomer, whereas hydrophobic contacts are made between both monomers (Fig. 2a). These interactions are conserved in both crystal forms. The isoalloxazine moiety of FMN interacts with residues involved in loops L7 and L11 of one monomer and loop L3′ of the other monomer. The two oxygen atoms of the isoalloxazine ring form hydrogen bonds with the main chain NH groups of the polypeptide moiety: O-2 with Gly-142, and O-4 with Asn-97 and Phe-98. O-4 also forms a hydrogen bond with Nϵ-2 of His-144. The nitrogen atoms of the isoalloxazine ring form hydrogen bonds with NH groups of the peptide moiety: N-1 with Gly-141, and N-5, which is thought to be the site of hydride transfer, with Asn-97. Although the redox potential of AzoR is unknown, the existence of the hydrogen bond donor to N5 of the isoalloxazine is attractive from the point of view of protein engineering, because this bond has been thought to directly affect the resonance characteristics of the pyridine ring that cause the alteration of redox properties in various flavoproteins (38.Fraaije M.W. Mattevi A. Trends Biochem. Sci. 2000; 25: 126-132Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). C7M of FMN makes hydrophobic interaction with Leu-50′. The isoalloxazine ring of FMN is close to planar (Fig. 2b). During data collection, the color of the crystal was yellow; therefore, this conformation is thought to be a fully oxidized one. The ribityl moiety interacts with residues involved in the β-strands β3 and β8 and loop L11. The OH groups of the ribityl moiety form hydrogen bonds with the polypeptide moiety. O-2* bonds the NH group of Gly-141 and the main chain carbonyl of Met-95. O-5* also forms a hydrogen bond with the side chain OH group of Ser-139. The phosphate group of FMN is anchored in a pocket formed by loop L1, the N-terminal region of helix α1, and β-strand β4. This phosphate group makes several hydrogen bonds with the polypeptide moiety. O1P bonds with the side chain OH groups of Ser-15 and Ser-17. The main chain NH group of Ser-17 also forms a hydrogen bond with O1P. O2P forms hydrogen bonds with the side chain OH groups of Ser-9 and Tyr-96, and O3P forms a hydrogen bond with the main chain NH group of Gln-16. The phosphate group of FMN is anchored in the pocket by hydrogen bonds rather than electrostatic interaction (Fig. 2c). Superposition of the Two Structures Obtained from the Different Crystal Forms—To investigate the conformational changes of the molecule, we superposed the two structures obtained from the different crystal forms (Fig. 3). The superposition demonstrated that the fold of the polypeptide chains and the environment of the cofactor FMN are extremely similar in each of the two structures, as mentioned above. The r.m.s. deviations for the positions of equivalent 197 Cα atoms and residues including side chains are 0.562 and 0.793 Å, respectively. However, the prominent conformational changes are found in three regions, which would mainly be a result of different crystal packing. The conformation of loop L4 is different between the P42212 and P4212 crystal structures. 3In this paragraph, primes for the distinction of positions in different monomers are omitted, because the two monomers are equivalent, and they need not be distinguished when referring not to one particular catalytic site. The flexibility of this region is deduced from the high average thermal factor (B factor) in both crystal forms. Residues 60-62 are particularly disordered in the P42212 crystal, and thus we could not build a model of this region. The second prominent conformational change is in loop L9, which covers the active site cavity. The largest shift of Cα in loop L9 is Asn-123, resulting in a 1.44 Å difference in position. In addition to loop L4, the flexibility of loop L9 is also deduced from the high B factor of the P4212 crystal structure. Although the B factor of loop L9 is relatively low in the P42212 crystal, it is attributed to the crystal packing in which loop L13 of the crystallographically related neighboring molecule penetrates the active site and tightly interacts with L9 (data not shown). The third important change is in the region that includes loop L13 and the N-terminal part of helix α6 (L13-Nα6). Pro-180, a residue included in the L13-Nα6 region, shows the largest change in the whole structure, resulting in a 3.05 Å difference in the Cα position. Structure Comparison with Other Flavoproteins—A structural similarity search in the Protein Data Bank with DALI (39.Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3565) Google Scholar) resulted in a number of matches to other flavoproteins, although they did not exhibit significant overall amino acid sequence similarity with AzoR. Proteins with a higher Z score than the value obtained for a typical prokaryote flavodoxin are as follows: the human FAD-dependent NAD(P)H:quinine oxidoreductase 1 (NQO1, originally called DT-diaphorase) (accession code 1D4A; Z score = 17.3 and r.m.s. deviation = 2.7 Å for 178 residues of a total 273 residues) (40.Faig 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 (177) Google Scholar), and the FAD-dependent rubredoxin:oxygen oxidoreductase (ROO) from Desulfovibrio gigas (accession code 1E5D; Z score = 13.2 and r.m.s. deviation = 2.3 Å for 135 residues of a total 401 residues) (41.Frazao 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. Biol. 2000; 11: 1041-1045Google Scholar). Bacillus subtilis Yhda protein (accession code 1NNI; Z score = 12.9 and r.m.s. deviation = 2.4 Å for 142 residues of a total 165 residues), although its function has not yet been documented, has a structure similar to that of yeast YLR011wp (accession code 1T0I; Z score = 19.4 and r.m.s. deviation = 2.3 Å for 156 residues of a total 191 residues, compared with 1NNI), and YLR001wp was characterized as an FMN-dependent NAD(P)H:ferric iron oxidoreductase (42.Liger D. Graille M. Zhou C.Z. Leulliot N. Quevillon-Cheruel S. Blondeau K. Janin J. van Tilbeurgh H. J. Biol. Chem. 2004; 279: 34890-34897Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). All of these flavoenzymes exist as homodimers. Fig. 4 shows a comparison of the topology and folding of these flavoenzymes with a ribbon diagram. In the structure of these enzymes, a core region, where a central five-stranded parallel β-sheet (Fig. 4, cyan) is flanked on each side by two and three helices (Fig. 4, purple), is well conserved (loops of this conserved region are drawn in green). However, there are some prominent differences in other parts. First, there is a difference with respect to the lengths of the regions corresponding to between β-strand β2 and helix α3 of AzoR, as well as the variance of the number of helices (Fig. 4, pink). These regions contribute to the formation of the active site cavities in all of these flavoproteins except ROO. Second, additional small C-terminal and N-terminal domains are found in NQO1 and ROO, respectively (Fig. 4, gray). The small C-terminal domain of NQO1 is responsible for the binding of the ADP moiety of NADP+, an analogue for electron donor NADPH (43.Li R. Bianchet M.A. Talalay P. Amzel L.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8846-8850Crossref PubMed Scopus (317) Google Scholar). The N-terminal domain of ROO, which has structural