Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M513345200 on May 9, 2006

J. Biol. Chem., Vol. 281, Issue 29, 20567-20576, July 21, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/29/20567    most recent
M513345200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ito, K.
Right arrow Articles by Tanokura, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ito, K.
Right arrow Articles by Tanokura, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Three-dimensional Structure of AzoR from Escherichia coli

AN OXIDEREDUCTASE CONSERVED IN MICROORGANISMS*

Kosuke Ito{ddagger}, Masayuki Nakanishi§, Woo-Cheol Lee{ddagger}, Hiroshi Sasaki{ddagger}, Shuhei Zenno, Kaoru Saigo, Yukio Kitade§, and Masaru Tanokura{ddagger}1

From the {ddagger}Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, the §Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan, and the Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received for publication, December 15, 2005 , and in revised form, April 5, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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). However, there is considerable concern about the toxicity, and especially the carcinogenicity, of some azo dyes (2). These compounds are frequently found in a chemically unchanged form even after wastewater treatment (3), 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, 5). 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) 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 (7-15). 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), Salmonella enterica (17), Photorhabdus luminescens (18), Vibrio vulnificus (19), Haemophilus influenzae (20), Pseudomonas putida (21), and so forth. On the other hand, AzoR has the ability to reduce menadione (vitamin K3, 2-methyl-1,4-naphthoquinone) (6). 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). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Protein Preparation and Crystallization—The recombinant E. coli AzoR used in this paper was expressed and purified as described previously (6). 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). 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 CAPS2 (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 x 0.6 x 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).

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).

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). Subsequently, maximum likelihood density modification was performed with RESOLVE (26), and the phases were further improved and extended to a resolution of 65.2-1.8 Å with ARP/wARP (27).

The initial structure in the P4212 crystal was determined by molecular replacement. The calculation of the molecular replacement was carried out with MOLREP (28) 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). 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). 2mFo - DFc and mFo - DFc SIGMAA-weighted electron density maps (31) 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). The superposition of molecules and calculation of the r.m.s. deviation between pairs of equivalent C{alpha} atoms and all atoms of proteins were executed using LSQKAB (33). Secondary structures were assigned using DSSP (34). Figure Preparation—The figures were prepared using PyMOL (35), LIGPLOT (36), and GRASP (37).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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). 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 beta-strands (beta1, beta2, beta3, beta8, and beta9) form an open twisted central beta-sheet surrounded on both sides by a total of six helices ({alpha}1-{alpha}6). Helices {alpha}1 and {alpha}6 are on one side of the beta-sheet, and helices {alpha}2, {alpha}3, {alpha}4, and {alpha}5 are located on the opposite side. The dimerization occurs mainly via anti-parallel side-to-side packing of the loop L7-helix {alpha}4 region and loop L11-helix {alpha}5 region of each monomer. The loop L3-helix {alpha}2 region also participates in the dimerization and interacts with the loop L3'-helix {alpha}2' 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.


View this table:
[in this window]
[in a new window]
  		TABLE 1
Statistics for data collection and heavy atom phasing

The values in parentheses are for the highest resolution shell.

 


View this table:
[in this window]
[in a new window]
  		TABLE 2
Refinement statistics

 


Figure 1
View larger version (61K):
[in this window]
[in a new window]
  		FIGURE 1.
Overall structure of AzoR and the secondary structure assignment. a, stereo view ribbon diagram of the homodimer of AzoR. The upper panel shows a view looking approximately along the molecular (crystallographic) 2-fold axis. The lower panel shows a molecule rotated ~90° from the upper panel. The two subunits of a molecule are colored yellow and green. Secondary structure assignments are labeled on the ribbon model. Cofactor FMN molecules are represented by a stick model, with carbon atoms in gray, oxygen atoms in red, nitrogen atoms in blue, and phosphorous atoms in orange. b, the diagram of the secondary structure elements for AzoR is shown on top of the amino acid sequence.

 
FMN Binding Site—The FMN prosthetic groups bind on the C-terminal end of the central beta-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 N{epsilon}-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). 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 beta-strands beta3 and beta8 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 {alpha}1, and beta-strand beta4. 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 C{alpha} 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.3 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 C{alpha} 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 {alpha}6 (L13-N{alpha}6). Pro-180, a residue included in the L13-N{alpha}6 region, shows the largest change in the whole structure, resulting in a 3.05 Å difference in the C{alpha} position.


Figure 2
View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 2.
Interactions between FMN and amino acid residues in the active site. a, schematic diagram showing contacts of the FMN cofactor to amino acid residues. Hydrogen bonds are shown as broken green lines (red residue numbers) and van der Waals' interactions by red shading (black residue numbers). Each atom element is represented by a sphere of different colors with a chemical symbol. b, SIGMAA-weighted 2mFo - DFc electron density maps surrounding the FMN. The map was calculated using the data of P42212 crystal structure and is contoured at 1.2 {sigma}. c, the electrostatic potential of AzoR is mapped onto the solvent-accessible surface, as calculated with GRASP. The FMN molecules shown in a and b are represented by a stick model, with color coding identical to that in Fig. 1.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Structure Comparison with Other Flavoproteins—A structural similarity search in the Protein Data Bank with DALI (39) 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), 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). Bacillus subtilis Yhda protein (accession code 1NNI [PDB] ; 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). 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 beta-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 beta-strand beta2 and helix {alpha}3 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). The N-terminal domain of ROO, which has structural similarity with beta-lactamase, reduces dioxygen to waters at the di-iron center, taking electrons from the FMN-containing, flavodoxin-like domain. Third, the structural functions of the regions corresponding to between helix {alpha}4 and beta-strand beta8 of AzoR are not equivalent (Fig. 4, yellow). This loop participates in the formation of the active site in AzoR and NQO1. On the other hand, this loop contributes to the stabilization of the additional N-terminal domain in ROO and contributes to the stabilization of the loop that is the component of the active site cavity in YLR011wp. Interestingly, these differences are found in the vicinity of the active site and probably result in the variety of the biochemical properties of these enzymes.


Figure 3
View larger version (65K):
[in this window]
[in a new window]
  		FIGURE 3.
Superposition of the structures from the two different crystal forms. a, the polypeptide moieties of the P42212 and P4212 crystal structures are represented as C{alpha} traces in blue and red, respectively. The FMN molecules are represented by a stick model, with the same color coding as for the polypeptide moieties. The three regions mentioned in the text are labeled to focus the one active site of the homodimer. b, a plot of the C{alpha} r.m.s. deviation between the P42212 and P4212 crystal structures. c, stereo view of the structures in the vicinity of the active site. The structures are represented by a stick model, with color coding identical to that in a except for FMN. Only the FMN of the P42212 structure is represented. Carbon, oxygen, nitrogen, and phosphorous atoms of FMN are colored in yellow, red, blue, and orange, respectively.

 
Our structural similarity search revealed that the flavoproteins, which share a similar structure with AzoR, are involved in various biological reactions. Among these enzymes, NQO1 is interesting because it has a function similar to that of AzoR regarding its substrate specificity. For example, both AzoR and NQO1 can exploit both methyl red and menadione as electron acceptors and NADH as electron donors (6, 44). NQO1 plays an important role in detoxification and exerts a protective effect against mutagenicity, carcinogenicity, and other toxicities via its reduction activity in the cytosol, which prevents the production of semiquinone intermediates (45-47). These facts imply that AzoR may also have an important role in detoxification in E. coli.


Figure 4
View larger version (46K):
[in this window]
[in a new window]
  		FIGURE 4.
The overall structure of AzoR and structurally homologous flavoproteins. Ribbon representation of AzoR (a), human NQO1 (b), ROO from D. gigas (c), and yeast YLR011wp (d). The color coding of the polypeptide moieties is described in detail in the text. Briefly, helices, strands, and loops of the conserved core region between four flavoproteins are colored purple, cyan, and green, respectively. The region between beta-strand beta2 and helix {alpha}3 of AzoR and the corresponding regions of the other flavoproteins are colored pink. The region between helix {alpha}4 and beta-strand beta8 of AzoR and the corresponding regions of the other flavoproteins are colored yellow. The flavin cofactors are represented by a stick model, and the isoalloxazine moieties of these molecules are indicated by arrows. Iron atoms in the beta-lactamase domain of ROO are shown by red spheres. The models in the lower panels are rotated by 90° along the horizontal axis compared with the upper panels for the flavoproteins.

 
Active Site and Conformational Changes—In the active site cavity, the re-face of FMN is fully occluded by a polypeptide moiety, and thus the reactions must occur at the si-face. In addition, there is not sufficient space near FMN to contact both the electron donor and acceptor simultaneously, which implies that only one substrate can bind to the enzyme at a time (Fig. 2c). This view is consistent with the ping-pong bi-bi mechanism for AzoR as has been proposed previously (6). Furthermore, the low average crystallographic B factors for FMN (Table 2), which are caused by the numerous contacts with amino acid residues mentioned above, suggest that FMN remains bound to the enzyme throughout the entire catalytic cycle.


Figure 5
View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 5.
Comparison of the active site of AzoR with that of NQO1. One active site is shown for each of AzoR (a) and rat NQO1 (b) (Protein Data Bank accession code 1QRD). The flavin cofactors, duroquinone, and the side chains of conserved amino acid residues in both enzymes are represented by a stick model, with carbon atoms in gray, oxygen atoms in red, nitrogen atoms in blue, and phosphorous atoms in orange. The polypeptide moieties of one subunit for each enzyme are drawn as C{alpha} traces in yellow and green.

 
Superposition of the two crystal structures revealed the prominent conformational changes in the molecule. Indeed, different crystal packing often affects the structure of inherently flexible region of a protein. In AzoR, loop L9' is positioned over the isoalloxazine ring moiety of FMN. The L13-N{alpha}6 also participates in the formation of the active site cavity. Therefore, it appears that these conformational changes are important for substrate binding or recognition, which can rearrange the structure of the active site cavity to accommodate substrates of different size. Loop L4' is ~15 Å away from the catalytic center (isoalloxazine ring) of the cofactor FMN. However, if the nicotinamide ring of NADH binds to the active site in an extended conformation, this loop could be close to the adenosine moiety of NADH, and thus loop L4' also may participate in substrate binding or recognition.

Implications for a Substrate Binding and Electron Transfer—To obtain direct information about the substrate binding, we tried to provide the crystal of AzoR that is complexed with substrates, substrate analogues, and competitive inhibitors by the soaking method. However, the electron density of these molecules could not be found, which is probably attributed to the crystal packing in which the enzymes stacked each other around the active site in both crystal forms, as mentioned above. We have also tried cocrystallization experiments and obtained some crystals, but it is not clear whether or not these enzymes are complexed with any compounds in these crystals at present. Instead, our results revealed that the locations of some amino acid residues around the isoalloxazine ring are conserved between AzoR and NQO1 (Fig. 5). Phe-98, Gly-141, Gly-142, Tyr-120', and Phe-162' of AzoR are found in the positions corresponding to Phe-106, Gly-149, Gly-150, Tyr-128', and Phe-178' in NQO1, respectively. Among these amino acid residues, Gly-142, Tyr-120', and Phe-162' of AzoR are attractive because the function of the corresponding amino acid residues in NQO1 have been clarified (40, 43). That is, the main chain NH group of Gly-150, which makes a hydrogen bond with O-4 of the isoalloxazine ring, forms a hydrogen bond with O-4' of the ribose adjacent to the nicotinamide of NADP+, a substrate analogue for the first half of the reaction of the ping-pong mechanism (electron donor).4 The side chain of Tyr-128' can adopt many different conformations. The different conformations result in opening and closing of the binding site, as well as in interactions with substrates of different size. Phe-178' stacks onto the nicotinamide ring of NADP+, resulting in sandwiching of the nicotinamide ring between the isoalloxazine ring and Phe-178'. Duroquinone (2,3,5,6-tetramethyl-quinone), a substrate for the second half of the reaction of the ping-pong mechanism (electron acceptor), is also sandwiched between the isoalloxazine ring and Phe-178'.If Phe-162' of AzoR stacks substrates in the manner of Phe-178' of NQO1, the configuration of a substrate and the isoalloxazine ring of a flavin cofactor would be ideal for a {pi}-stacking system (48) and direct hydride transfer (43). Menadione, which has also been reported as an electron acceptor for AzoR, occupies an area corresponding to between Phe-162' and the isoalloxazine ring of AzoR in QR2 (quinone reductase type 2), the homologous protein of NQO1 in mammals (49). These observations suggest that Gly-142, Tyr-120', and Phe-162' of AzoR may play similar roles in the catalysis. Furthermore, the conservation of these amino acid residues in orthologues of the AzoR protein lends further support to our propositions. Indeed, binding of substrates by means of a {pi}-stacking system using phenylalanine in the vicinity of an isoalloxazine ring is often observed in the crystal structures of flavoproteins (50-55). Hence, the aromatic ring of azo compounds may also be stacked on Phe-162' of AzoR using a {pi}-stacking system, with their azo group laid almost on top of N-5 of the isoalloxazine ring for an effective hydride transfer.

The C-terminal domain of NQO1 is responsible for the binding of the ADP moiety of NADP+ (43, 56). As mentioned above, the part corresponding to the C-terminal domain of NQO1 is lacking in AzoR. In addition, AzoR does not contain an NADH-binding motif (GXGXXG). These facts strongly suggest that NADH binds to the AzoR in a different manner than NQO1 and that AzoR may have a novel NADH-binding mechanism.

Generally, two electron-reduced FMN is protonated at N-1 of isoalloxazine ring to compensate for the negative charge built by hydride transfer. In AzoR, however, no functional groups can donate a proton to the N-1 position. Therefore, an enol tautomer at the O-2 position of isoalloxazine ring is proposed as the alternative site of protonation, in which proton is donated from N{epsilon}-2 of His-144 to the O-2 of isoalloxazine (Fig. 2a). The other possibility is that the negative charge is delocalized at the N-1-C-2=O-2 locus, and it is stabilized by the positive charge of imidazole ring of His-144. These states of isoalloxazine are feasible from a standpoint on a research of molecular orbital (57).

In conclusion, we have determined the three-dimensional structure of AzoR from E. coli in two different crystal forms. AzoR would likely act as a homodimeric FMN-containing enzyme and has a flavodoxin-like structure without the explicit overall amino acid sequence homology. Superposition of the two structures from different crystal forms revealed the regions that allow conformational change near the active site. Comparison of the active site with NQO1 suggests some of the amino acid residues that may be involved in substrate binding. These amino acid residues are conserved in orthologues of the AzoR protein. It is also suggested that AzoR has a novel NADH-binding mechanism. The structure of AzoR will provide essential clues for the elucidation of the molecular mechanism of its function, as well as that of AzoR orthologues and the development of the biodegradation system.


   FOOTNOTES
 
The atomic coordinates and structure factors (codes 1v4b and 2d5i) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported in part by the National Project on Protein Structural and Functional Analyses of the Ministry of Education, Culture, Sports, Science and Technology of Japan and by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The synchrotron-radiation experiments were performed at BL6A and BL18B in PF (Tsukuba, Japan) and BL41XU in SPring-8 (Harima, Japan) with the approval of the Photon Factory, KEK (Proposals 2000G307 and 2003G127), and the Japan Synchrotron Radiation Research Institute (Proposal 2002A0741-RL1-np), respectively. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 81-3-5841-5165; Fax: 81-3-5841-8023; E-mail: amtanok{at}mail.ecc.u-tokyo.ac.jp.

2 The 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. Back

3 In 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. Back

4 The three-dimensional structure of rat NQO1 complexed with NADP+ has been reported (43). However, the coordinates have not been deposited in the Protein Data Bank. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Meyer, U. (1981) FEMS Symp. 12, 371-385
  2. Holme, I. (1984) Ecological Aspects of Color Chemistry: Developments in the Chemistry and Technology of Organic Dyes (Griffiths, J., ed) pp. 111-128, Society of Chemistry Industry, Oxford
  3. Levine, W. G. (1991) Drug Metab. Rev. 23, 253-309[Medline] [Order article via Infotrieve]
  4. Robinson, T., McMullan, G., Marchant, R., and Nigam, P. (2001) Bioresour. Technol. 77, 247-255[CrossRef][Medline] [Order article via Infotrieve]
  5. Stolz, A. (2001) Appl. Microbiol. Biotechnol. 56, 69-80[CrossRef][Medline] [Order article via Infotrieve]
  6. Nakanishi, M., Yatome, C., Ishida, N., and Kitade, Y. (2001) J. Biol. Chem. 276, 46394-46399[Abstract/Free Full Text]
  7. Zimmermann, T., Kulla, H. G., and Leisinger, T. (1982) Eur. J. Biochem. 129, 197-203[Medline] [Order article via Infotrieve]
  8. Zimmermann, T., Gasser, F., Kulla, H. G., and Leisinger, T. (1984) Arch. Microbiol. 138, 37-43[CrossRef][Medline] [Order article via Infotrieve]
  9. Ghosh, D. K., Mandal, A., and Chaudhuri, J. (1992) FEMS Microbiol. Lett. 77, 229-233[CrossRef][Medline] [Order article via Infotrieve]
  10. Ghosh, D. K., Ghosh, S., Sadhukhan, P., Mandal, A., and Chaudhuri, J. (1993) Indian J. Exp. Biol. 31, 951-954[Medline] [Order article via Infotrieve]
  11. Rafii, F., and Cerniglia, C. E. (1993) Appl. Environ. Microbiol. 59, 1731-1734[Abstract/Free Full Text]
  12. Suzuki, Y., Yoda, T., Ruhul, A., and Sugiura, W. (2001) J. Biol. Chem. 276, 9059-9065[Abstract/Free Full Text]
  13. Moutaouakkil, A., Zeroual, Y., Zohra Dzayri, F., Talbi, M., Lee, K., and Blaghen, M. (2003) Arch. Biochem. Biophys. 413, 139-146[CrossRef][Medline] [Order article via Infotrieve]
  14. Maier, J., Kandelbauer, A., Erlacher, A., Cavaco-Paulo, A., and Gubitz, G. M. (2004) Appl. Environ. Microbiol. 70, 837-844[Abstract/Free Full Text]
  15. Ramalho, P. A., Paiva, S., Cavaco-Paulo, A., Casal, M., Cardoso, M. H., and Ramalho, M. T. (2005) Appl. Environ. Microbiol. 71, 3882-3888[Abstract/Free Full Text]
  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., and Garcia, E. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 13826-13831[Abstract/Free Full Text]
  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., and Wilson, R. K. (2004) Nat. Genet. 36, 1268-1274[CrossRef][Medline] [Order article via Infotrieve]
  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., and Kunst, F. (2003) Nat. Biotechnol. 21, 1307-1313[CrossRef][Medline] [Order article via Infotrieve]
  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., and Tsai, S. F. (2003) Genome Res. 13, 2577-2587[Abstract/Free Full Text]
  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., and Munson, R. S., Jr. (2005) J. Bacteriol. 187, 4627-4636[Abstract/Free Full Text]
  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., and Fraser, C. (2002) Environ. Microbiol. 4, 799-808[CrossRef][Medline] [Order article via Infotrieve]
  22. Unden, G., and Bongaerts, J. (1997) Biochim. Biophys. Acta 1320, 217-234[Medline] [Order article via Infotrieve]
  23. Ito, K., Nakanishi, M., Lee, W. C., Sasaki, H., Zenno, S., Saigo, K., Kitade, Y., and Tanokura, M. (2005) Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 61, 399-402[CrossRef]
  24. Collaborative Computational Project, Number 4 (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760-763[CrossRef][Medline] [Order article via Infotrieve]
  25. Terwilliger, T. C., and Berendzen, J. (1999) Acta Crystallogr. Sect. D Biol. Crystallogr. 55, 849-861[CrossRef][Medline] [Order article via Infotrieve]
  26. Terwilliger, T. C. (2000) Acta Crystallogr. Sect. D Biol. Crystallogr. 56, 965-972[CrossRef][Medline] [Order article via Infotrieve]
  27. Perrakis, A., Morris, R., and Lamzin, V. S. (1999) Nat. Struct. Biol. 6, 458-463[CrossRef][Medline] [Order article via Infotrieve]
  28. Vagin, A., and Teplyakov, A. (1997) J. Appl. Crystallogr. 30, 1022-1025[CrossRef]
  29. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Acta Crystallogr. Sect. D Biol. Crystallogr. 53, 240-255[CrossRef][Medline] [Order article via Infotrieve]
  30. McRee, D. E. (1999) J. Struct. Biol. 125, 156-165[CrossRef][Medline] [Order article via Infotrieve]
  31. Read, R. J. (1986) Acta Crystallogr. Sect. A 42, 140-149[CrossRef]
  32. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26 283-291[CrossRef]
  33. Kabsch, W. (1976) Acta Crystallogr. Sect. A 32, 922-923[CrossRef]
  34. Kabsch, W., and Sander, C. (1983) Biopolymers 12, 2577-2637
  35. DeLano, W. L. (2002) The PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, CA
  36. Wallace, A. C., Laskowski, R. A., and Thornton, J. M. (1995) Protein Eng. 8, 127-134[Abstract/Free Full Text]
  37. Nicholls, A., Sharp, K. A., and Honig, B. (1991) Proteins 11, 281-296[CrossRef][Medline] [Order article via Infotrieve]
  38. Fraaije, M. W., and Mattevi, A. (2000) Trends Biochem. Sci. 25, 126-132[CrossRef][Medline] [Order article via Infotrieve]
  39. Holm, L., and Sander, C. (1993) J. Mol. Biol. 233, 123-138[CrossRef][Medline] [Order article via Infotrieve]
  40. Faig, M., Bianchet, M. A., Talalay, P., Chen, S., Winski, S., Ross, D., and Amzel, L. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 3177-3182[Abstract/Free Full Text]
  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., and Le Gall, J. (2000) Nat. Struct. Biol. 11, 1041-1045
  42. Liger, D., Graille, M., Zhou, C. Z., Leulliot, N., Quevillon-Cheruel, S., Blondeau, K., Janin, J., and van Tilbeurgh, H. (2004) J. Biol. Chem. 279, 34890-34897[Abstract/Free Full Text]
  43. Li, R., Bianchet, M. A., Talalay, P., and Amzel, L. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8846-8850[Abstract/Free Full Text]
  44. Bayney, R. M., Rodkey, J. A., Bennett, C. D., Lu, A. Y., and Pickett, C. B. (1987) J. Biol. Chem. 262, 572-575[Abstract/Free Full Text]
  45. Smith, M. T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 7624-7626[Free Full Text]
  46. Benson, A. M., Hunkeler, M. J., and Talalay, P. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5216-5220[Abstract/Free Full Text]
  47. Chesis, P. L., Levin, D. E., Smith, M. T., Ernster, L., and Ames, B. N. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1696-1700[Abstract/Free Full Text]
  48. McGaughey, G. B., Gagne, M., and Rappe, A. K. (1998) J. Biol. Chem. 273, 15458-15463[Abstract/Free Full Text]
  49. Foster, C. E., Bianchet, M. A., Talalay, P., Zhao, Q., and Amzel, L. M. (1999) Biochemistry 38, 9881-9886[CrossRef][Medline] [Order article via Infotrieve]
  50. Lovering, A. L., Hyde, E. I., Searle, P. F., and White, S. A. (2001) J. Mol. Biol. 309, 203-213[CrossRef][Medline] [Order article via Infotrieve]
  51. Haynes, C. A., Koder, R. L., Miller, A. F., and Rodgers, D. W. (2002) J. Biol. Chem. 277, 11513-11520[Abstract/Free Full Text]
  52. Koike, H., Sasaki, H., Kobori, T., Zenno, S., Saigo, K., Murphy, M. E., Adman, E. T., and Tanokura, M. (1998) J. Mol. Biol. 280, 259-273[CrossRef][Medline] [Order article via Infotrieve]
  53. Karplus, P. A., and Schulz, G. E. (1989) J. Mol. Biol. 210, 163-180[CrossRef][Medline] [Order article via Infotrieve]
  54. Deng, Z., Aliverti, A., Zanetti, G., Arakaki, A. K., Ottado, J., Orellano, E. G., Calcaterra, N. B., Ceccarelli, E. A., Carrillo, N., and Karplus, P. A. (1999) Nat. Struct. Biol. 6, 847-853[CrossRef][Medline] [Order article via Infotrieve]
  55. Ziegler, G. A., Vonrhein, C., Hanukoglu, I., and Schulz, G. E. (1999) J. Mol. Biol. 289, 981-990[CrossRef][Medline] [Order article via Infotrieve]
  56. Zhao, Q., Yang, X. L., Holtzclaw, W. D., and Talalay, P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1669-1674[Abstract/Free Full Text]
  57. Cavelier, G., and Amzel, L. M. (2001) Proteins 43, 420-432[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 MicrobiologyHome page
H. Chen, H. Xu, O. Kweon, S. Chen, and C. E. Cerniglia
Functional role of Trp-105 of Enterococcus faecalis azoreductase (AzoA) as resolved by structural and mutational analysis
Microbiology, September 1, 2008; 154(9): 2659 - 2667.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Ito, M. Nakanishi, W.-C. Lee, Y. Zhi, H. Sasaki, S. Zenno, K. Saigo, Y. Kitade, and M. Tanokura
Expansion of Substrate Specificity and Catalytic Mechanism of Azoreductase by X-ray Crystallography and Site-directed Mutagenesis
J. Biol. Chem., May 16, 2008; 283(20): 13889 - 13896.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/29/20567    most recent
M513345200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ito, K.
Right arrow Articles by Tanokura, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ito, K.
Right arrow Articles by Tanokura, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement