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Originally published In Press as doi:10.1074/jbc.M500666200 on March 16, 2005

J. Biol. Chem., Vol. 280, Issue 22, 21144-21154, June 3, 2005
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Crystal Structure of the Hydroxyquinol 1,2-Dioxygenase from Nocardioides simplex 3E, a Key Enzyme Involved in Polychlorinated Aromatics Biodegradation*

Marta Ferraroni{ddagger}, Jana Seifert§, Vasili M. Travkin¶, Monika Thiel§, Stefan Kaschabek§, Andrea Scozzafava{ddagger}, Ludmila Golovleva¶, Michael Schlömann§, and Fabrizio Briganti{ddagger}||

From the {ddagger}Dipartimento di Chimica, Università di Firenze, Via della Lastruccia 3, Sesto Fiorentino I-50019, Italy, the §TU Bergakademie Freiberg Interdisziplinäres Ökologisches Zentrum, Freiberg D-09599, Germany, and the Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino Moscow Region 142290, Russia

Received for publication, January 19, 2005 , and in revised form, March 10, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Hydroxyquinol 1,2-dioxygenase (1,2-HQD) catalyzes the ring cleavage of hydroxyquinol (1,2,4-trihydroxybenzene), a central intermediate in the degradation of aromatic compounds including a variety of particularly recalcitrant polychloro- and nitroaromatic pollutants. We report here the primary sequence determination and the analysis of the crystal structure of the 1,2-HQD from Nocardioides simplex 3E solved at 1.75 Å resolution using the multiple wavelength anomalous dispersion of the two catalytic irons (1 Fe/293 amino acids). The catalytic Fe(III) coordination polyhedron composed by the side chains of Tyr164, Tyr197, His221, and His223 resembles that of the other known intradiol-cleaving dioxygenases, but several of the tertiary structure features are notably different. One of the most distinctive characteristics of the present structure is the extensive openings and consequent exposure to solvent of the upper part of the catalytic cavity arranged to favor the binding of hydroxyquinols but not catechols. A co-crystallized benzoate-like molecule is also found bound to the metal center forming a distinctive hydrogen bond network as observed previously also in 4-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus 1CP. This is the first structure of an intradiol dioxygenase specialized in hydroxyquinol ring cleavage to be investigated in detail.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Hydroxyquinol (1,2,4-trihydroxybenzene) (HQ)1 is one of the central intermediates in the degradation of a large variety of aromatic compounds. It has been detected in the breakdown of 4-hydroxybenzoate, resorcinol, salicylate, vanillate, benzoate, protocatechuate, and gentisate, by fungi such as Trichosporon cutaneum or Phanerochaete chrysosporium (1–3). In bacteria HQ has been identified as an intermediate in the degradation of mononuclear hydroxyaromatic compounds such as resorcinol and 2,4-dihydroxybenzoate (4, 5) or amino-hydroxyaromatic compounds such as 4-aminophenol (6) as well as in the degradation of hydroxylated biaryl ethers such as 2-hydroxydibenzo-p-dioxin and 3-hydroxydibenzofuran (7). HQ also occurs in the catabolic pathways of aromatic compounds carrying nitro groups such as in 3- and 4-nitrophenol or as 4-nitrocatechol (8–12).

HQ and its chloro-substituted derivatives 5-chlorohydroxyquinol (5CHQ) and 6-chlorohydroxyquinol (6CHQ) play an especially important role in the bacterial degradation of phenols or phenoxyacetates carrying a chloro-substituent in para position to the OH or OCH2COO– group, respectively. Thus, pentachlorophenol by rhodococci and mycobacteria has been reported to be degraded via HQ (13–16), whereas in Sphingobium chlorophenolicum (Sphingomonas chlorophenolica) already the 2,6-dichloroquinol appears to be subject to ring cleavage and, in contrast to earlier reports, no 6CHQ is formed (17). On the contrary, for 2,4,6-trichlorophenol breakdown 6CHQ has been suggested as an intermediate for Streptomyces rochei 303 as well as several Gram-negative bacteria (18–24). 2,6-Dichlorophenol can also be degraded via 6CHQ, whereas 2,4-dichlorophenol, 4- and 2-chlorophenol by 2,4,6-trichlorophenol-induced cells may be transformed on the same pathway, but yielding HQ as a ring cleavage substrate (18, 19, 24). In 2,4,5-trichlorophenoxyacetate degradation by Burkolderia (Pseudomonas) cepacia AC1100 5CHQ and HQ are formed sequentially as intermediates (25). Although 3,5-dichlorohydroxyquinol was found to be an intermediate of 2,4-dichlorophenoxyacetate degradation by Nocardioides simplex 3E, HQ may also be involved as a ring cleavage substrate (26, 27).

HQs are degraded aerobically by specialized intradiol ring-cleaving dioxygenases; the most studied enzymes from this family are the protocatechuate 3,4-dioxygenases (3.4-PCDs), the catechol 1,2-dioxygenases (1,2-CTDs), and the chlorocatechol 1,2-dioxygenases (1,2-CCDs) which generally possess distinctive substrate specificities (28). The hydroxyquinol 1,2-dioxygenases (1,2-HQDs hereafter) catalyze the intradiol cleavage of hydroxyquinols to form 3-hydroxy-cis,cis-muconates, which occur in solution in the keto form, i.e. as maleylacetate (Scheme 1) (29).

Several 1,2-HQDs have been purified and characterized from a variety of microorganisms such as Gram-negative bacteria (B. cepacia AC1100, Azotobacter sp. GP1, Ralstonia pickettii DTP0602, Burkholderia sp. strain AK-5), Gram-positive bacteria (S. rochei 303, N. simplex 3E, Arthrobacter sp. strain BA-5-17) and also from fungi (T. cutaneum, P. chrysosporium), but very little is known about the factors controlling substrate specificity for this novel group of intradiol dioxygenases (1, 2, 6, 20, 24, 25, 30–32). 1,2-HQD from N. simplex 3E is a homodimer with a molecular weight of about 65,000, containing Fe(III) ions essential for its activity with quaternary structure ({alpha} Fe(III))2 (30). X-ray absorption spectroscopy studies showed that in the native enzyme as well as in the enzyme-substrate complex, the iron is pentacoordinated, with an average Fe-L distance of 1.93 Å and that histidines are present in the metal coordination sphere (2, 33).



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  		SCHEME 1
 
To date, structural information is available only for a few intradiol dioxygenases from the 3,4-PCD and the 1,2-CTD families. The 3,4-PCD family has been studied extensively with enzymes from Pseudomonas aeruginosa, and Acinetobacter "calcoaceticus" ADP1; their adducts with substrates and inhibitors were also characterized (34–40). For the 1,2-CTD family the structures of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus ADP1 (Ac 1,2-CTD hereafter) and 4-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus 1CP (Rho 1,2-CCD) have been solved recently (41, 42).

DNA sequencing showed that 1,2-HQDs are most closely related to catechol and chlorocatechol dioxygenases (7, 11, 21, 31, 32, 43). Nevertheless, 1,2-HQDs appear to have a distinct substrate specificity and do not, or relatively slowly, convert catechol or substituted catechols, respectively (1, 2, 6, 7, 20, 22, 24, 25, 30–32). Because 1,2-HQDs on one hand and (chloro)catechol dioxygenases on the other belong to different catabolic pathways, and correspondingly, the respective genes belong to different operons, the development of HQD substrate specificity was a very important step in the evolution of pathways for the efficient biodegradation of natural aromatic compounds as well as of xenobiotics. The analysis of the first crystal structure of a 1,2-HQD from N. simplex 3E (hereafter Ns 1,2-HQD), an enzyme that catalyzes the degradation of HQ with markedly high selectivity (30), could shed some light on the structural factors governing substrate specificity in a group of enzymes that catalyze key reactions in the biodegradation of toxic compounds.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Strain, Protein Preparation, and Sequencing of N Terminus and Peptides—Growth of the 2,4,5-trichlorophenoxyacetic acid utilizing strain N. simplex 3E with 2,4-dichlorophenoxyacetic acid as an alternative carbon source has been reported previously (44). 1,2-HQD from N. simplex 3E was purified as reported previously (30). Tryptic peptides were isolated as described for other enzymes (45, 46) using 1 mg of 1,2-HQD for the digestion. Sequencing of the N terminus and of tryptic peptides was performed using an Applied Biosystems model 473A sequencer.

Cloning and Sequencing of the 1,2-HQD Gene—General methods for isolation and manipulation of DNA and cultivation of Escherichia coli cells were as reported previously (47). pBluescript II SK(+) obtained from Stratagene was used as general cloning vector, and a T vector (48) derived from it was used for cloning PCR products. Recombinant plasmids were transformed into E. coli DH5{alpha}, bought from Invitrogen. Genomic DNA from N. simplex 3E was prepared by the method of Wilson (49).

Primers for the amplification of the 1,2-HQD gene were designed for conserved regions of the 1,2-HQDs of R. pickettii (hadC), Arthrobacter sp. strain BA-5-17, Sphingomonas wittichii RW1 (dxnF), B. cepacia (tftH), and Agrobacterium tumefaciens C58 (7, 31, 32, 43). Primer HQD-fw1 (5'-CGS CAG GAR TKS ATC CTG-3') targets the bases corresponding to amino acid positions 82–87 in the alignment (see Fig. 4), whereas primer HQD-rev1 (5'-CCR TCR KNM GGN ATS GGR TA-3') is expected to bind to the bases corresponding to positions 219–224 in the alignment (see Fig. 4). Thus, the PCR products had an expected length of about 400 bp.

The PCR mixture (50 µl) contained 30 pmol of each primer, 0.5 µgof genomic template DNA, 20 µM each deoxynucleotide triphosphate, 1 x PCR buffer (MBI Fermentas), 1.0 unit of DNA Taq polymerase (MBI Fermentas), 1.5 mM MgCl2, 5% dimethyl sulfoxide, and 0.5% bovine serum albumin. The PCR was performed with a touchdown thermocycle program: an initial denaturation (95 °C, 5 min); 10 cycles with decreasing annealing temperature (60–50 °C, 30 s), polymerization (72 °C, 1 min), and denaturation (95 °C, 30 s); 20 more cycles with 50 °C as the annealing temperature; and an additional 5 min of polymerization during the last cycle.

After cloning of the 400-bp PCR product into a T vector, giving rise to plasmid pNocSi01, sequencing of the fragment proved it to be homologous to the corresponding segments of other 1,2-HQD genes. Labeling of the 400-bp fragment by a DIG DNA Labeling and Detection Kit Non-radioactive (Roche Applied Science) was performed as described in the Roche manual. The probe was then used to detect the corresponding fragment on a Southern blot of 0.64 µg of N. simplex 3E DNA digested with BamHI, PstI, SacI, and XhoI, respectively, and run on a 1% agarose gel with 1*TAE buffer (47). From a second gel, an area that corresponded in size to the hybridization signal (3 kb, SacI) was excised, and the included DNA was eluted and ligated into the dephosphorylated SacI site of pBluescript II SK(+). After transformation of the ligation mixture into E. coli DH5{alpha}, the labeled insert of pNocSi01 was used to identify clone pNocSi89 by colony hybridization.

The nucleotide sequence of the 1,2-HQD was determined by preparing subclones of pNocSi89 with the restriction enzymes SacII and XhoI. Two different 650-bp SacII restriction fragments and a 1.2-kb XhoI fragment, respectively, encode the complete sequence of the gene. For sequencing reactions, a MBI Fermentas CycleReader Auto DNA Sequencing Kit was used, with subsequent electrophoresis with a Li-cor 4200 IR2 sequencer and analysis with the e-Seq program (version 1.2). Sequences were assembled using Staden Package version 2002.0. The sequence is available under GenBank/EMBL/DDBJ accession number AY822041 [GenBank] . Comparisons with data-base entries were performed by using BLASTX (50). Multiple sequence alignments were created using ClustalX (version 1.8) (51).

Crystallization and Data Collection—The enzyme was crystallized at 293 K using the sitting drop vapor diffusion method from a solution containing 2.0 M ammonium sulfate, 4% polyethylene glycol 400, 100 mM Hepes pH 7.5 (52). The drops consisted of 4 µl of 20 mg/ml protein solution and 6 µl of reservoir solution equilibrated against 50 µl of reservoir solution (Crystal Clear Strips from Molecular Dimension, Inc.).

A native data set extending to a maximum resolution of 1.75 Å was collected at the X11 beamline, EMBL, DESY, Hamburg. Data were collected using a MAR CCD165 detector at a wavelength of 0.908 Å. Crystals belong to the primitive monoclinic space group P21 with unit cell dimensions a = 46.28, b = 84.98, c = 83.92 Å, {beta} = 92.84°. For all data collections crystals of the native enzyme were cooled at 100 K adding 17% ethylene glycol to the mother liquor solution as cryoprotectant. Crystals suffered from damage if they were transferred in solution different from their mother solution unless they were previously cross-linked adding glutaraldehyde to the drops up to a final concentration of roughly 2% (v/v).

Metal Content Analysis—Analysis of the protein metal content was performed by using a PerkinElmer Optima 2000 Inductively Coupled Plasma AES (Atomic Emission Spectrometry) Dual Vision. The metal content analysis revealed the presence of 2 equivalents of iron ions and 1 equivalent of copper ions/mol of protein.

Structure Determination and Refinement—All molecular replacements attempts, using coordinates of known intradiol dioxygenases structures as a model, failed to provide a solution for Ns 1,2-HQD.

The structure of the enzyme was, therefore, solved by multiple wave-length anomalous dispersion (MAD) using the anomalous signal of the two catalytic irons. MAD data were collected at the BM14 beamline, ESFR, Grenoble. The data collected at three wavelengths (inflection, peak, remote) were processed and integrated with DENZO and scaled by SCALEPACK, from the HKL program suite (53).

The program SOLVE (54) was used to identify the two iron sites and for phase calculation. The 2.6 Å MAD phases were improved and extended to 2.2 Å by solvent flattening and histogram mapping using the program DM from the CCP4 program suite (55). Automatic tracing was performed initially with the program RESOLVE (56) and extended using ARP/WARP version 6.0 (57). After 200 cycles of refinement and 20 cycles of autobuilding, 532 amino acids of 586 were found and placed in 16 chains with a global connectivity index of 0.94. After this process manual intervention was required to complete the model. The model was initially refined against 2.2 Å resolution (MAD remote wavelength data set) and finally against 1.75 Å data, using the program Refmac 5.1.24 from the CCP4 program suite (55). Manual rebuilding of the model was performed using the program QUANTA (58). Solvent molecules were introduced automatically using ARP (57). Refinement resulted in R factor and Rfree values of 19.2 and 24.6%, respectively. Data processing and refinement statistics are summarized in Table I. The overall mean B factor of the structure after refinement was 25.68 Å2 for chain A, 28.23 Å2 for chain B, and 29.15 Å2 for all atoms.


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  		TABLE I
Summary of data collection and atomic model refinement statistics

 
Protein coordinates have been deposited in the Protein Data Bank (accession number 1TMX [PDB] ).

The final model is composed of residues 2–293 for chain A and 4–293 for chain B, two Fe(III) ions, two benzoate ions, two phospholipids (C13/C17), two sulfate ions, one copper ion, one chloride ion, and 837 water molecules.

There are two disordered regions in chain B corresponding to residues 67–72 and 263–275. Electron density was missed for residues B264, B265, and B270 even for the main chain atoms, and they were not introduced in the model. Furthermore, no electron density was visible for the side chains of residues: A2 Ser, A73 Glu, B8 Glu, B71 Thr, B72 Asn, B75 Arg, B95 Asn, B264 Arg, B266 Pro, B276 Gln, and B277 Ile.

Double conformations for the side chains of residues A118 Arg, A155 Val, A195 Lys, A222 Leu, A244 Glu, A247 Asp, A285 Arg were modeled.

A spheric electron density in the Fo – Fc map at 12{sigma} was found bound to His A42 (2.12 Å) and His B42 (2.02 Å), and it could be easily attributed to a metal ion. A peak at lower height, compared with the iron ones, was present in the Harker section of the anomalous Patterson maps corresponding to the position of the metal ion. The metal should be one close to the iron as number of electrons because of the height of the peak in the Fo – Fc map (assuming a unitary occupancy) and because it should have an absorption edge near to the iron K edge. The coordination sphere of the metal is completed by a chloride ion (2.57 Å).

An electron density was found close to the iron in both active sites, and it was explained as a benzoate-like molecule bound to the iron in a bidentate way.

Two sulfate ions were found on the surface of the A chain, hydrogen bonded to some water molecules and side chain atoms.

Structure Analysis—The stereochemical quality of the models was assessed using the program PROCHECK (59). The Ramachandran plot is of a good quality, with 479 non-glycine and non-proline residues; among these, 438 (91.4%) are in the most favored regions, 38 (7.9%) are in the additional allowed regions, 1 (2%) (Thr B71) is in the generously allowed regions, and 2 (4%) (Glu B73 and Arg B74) are in disallowed regions.

The secondary structure was defined utilizing the DSSP data base and program (60).

Global structure superimpositions were carried out by utilizing the matching algorithm implemented into the HEX 4.2 program (61). Least squares fits of the active site regions were performed using the McLachlan algorithm as implemented in the program ProFit 2.2 (www.bioinf.org.uk/software/profit/) specifying as the fitting subset the four amino acid ligands to the catalytic iron ions (62).

Electrostatic Potentials were estimated first transforming the protein data base coordinate file into a pqr file containing partial charges and radii for each atom by using the PDB2PQR web service and then solving the second order differential Poisson-Boltzmann equation, which relates the electrostatic potential in a dielectric to the charge density using the macroscopic electrostatics with atomic details (MEAD) program package (63, 64).

HQ was docked manually into the active site by first simulating the dissociation of Tyr197 from the iron center. It was assumed that the substrates bind to the iron in a bidentate fashion and with orientations of their aromatic ring similar to those observed for catechol or 4-methylcatechol in Ac 1,2-CTD or for the benzoate ion in Ns 1,2-HQD and Rho 1,2-CCD (41, 42). Slight rotations and/or tilts of the iron-bound substrate molecules did not result in changes in the amino acid residues interacting with the substrates ring substituents.

PyMol, UCSF Chimera, and MS/MS were used to produce ribbon diagrams, electrostatic potential surfaces, electron density and other representations (65–67).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Gene and Protein Sequence—Using primers directed toward regions conserved in other 1,2-HQDs, it was possible to amplify a segment of the expected size (400-bp) from genomic DNA of N. simplex 3E. The use of this fragment as a probe allowed us to clone a 3-kbp fragment of N. simplex 3E DNA into E. coli. Sequencing of two 650-bp and a 1.2-kbp region from several subclones resulted in the complete sequence of the presumed 1,2-HQD gene.

Independently from the genetic approach, the following sequences were obtained by amino acid sequencing of the N terminus and of nine tryptic peptides of the purified protein (amino acids separated by slashes or X indicating uncertain positions): 1,2-HQD-27 (S/X A A/D S/X LN S/X), 1,2-HQD-36 (SFDATADPR X/R), 1,2-HQD-51 (A/E I T/D P/G TP), 1,2-HQD-55 (IESGGDI), 1,2-HQD-61/63 (IEV W/X EADDDGFY D/X VQYDD D/X), 1,2-HQD-70 (A/L T/H E/L A/L E/S), 1,2-HQD-72 (TLVTXIF M/F), 1,2-HQD-77 (RQEFILL). All of these peptides, except 1,2-HQD-27 (for which the sequence was of low quality), occur in the sequence predicted from the cloned N. simplex 3E gene (as well as in those from the tftH, hadC, dxnF genes), thus proving that, in fact, the gene of a 1,2-HQD was cloned and sequenced.

The most similar sequence in the data base was that of 1,2-HQD from Arthrobacter sp. strain BA-5-17 (32) (73% identical positions in the alignment of Fig. 4). The similarity to other 1,2-HQDs ranged from 42 to 69% identical positions (see alignment of Fig. 4). In contrast, the similarity to the representatives of catechol and chlorocatechol 1,2-dioxygenases given in Fig. 4 was between 22 and 30% identical positions.



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  		FIG. 1.
Schematic representation of the overall structure of Ac 1,2-CTD (A), Ns 1,2-HQD (B), 90° rotated B around the horizontal axis (C). The iron and copper ions are represented as blue and magenta spheres, respectively; the phospholipid molecules are shown in yellow.

 
Overall Structure and Linker Domain—The 1,2-HQD from the Gram-positive bacterium N. simplex 3E is a homodimeric protein with overall dimensions 110 x 50 x 50 Å. The statistics for data collection, phasing, and structure refinement are summarized in Table I. The final model is composed of residues 2–293 for chain A and 4–293 for chain B, two Fe(III) ions, one copper ion, two benzoate ions, two phospholipids (C13/C17), two sulfate ions, one chloride ion and 837 water molecules.

The general topology of Ns 1,2-HQD resembles that of Ac 1,2-CTD and comprises two catalytic domains separated by a common "{alpha}-helical zipper" motif that consists of six N-terminal helices from each subunit.

Two phospholipid molecules are located inside a large hydrophobic channel formed by the two protein monomers at the interface between the two subunits and in the center of the linker domain, with the head group directed outward into the solvent and the tail moieties pointing inward, toward each other (Fig. 1). A phosphatidylcholine molecule with two C12–13/C17 hydrophobic tails was used as a model because the absence of the electron density of the head groups did not allow determination of their precise identity, and the length of each tail was based on the length of the electron density and on the stereochemistry of known phospholipids. The presence of such phospholipids appears to be distinctive for this class of enzymes, although their possible role has still to be clarified (41, 42).

The linker domain is mainly composed of three long (H1–H3) and three short (H4–H6) {alpha}-helices supplied by each subunit: five helices from the N terminus of each monomer are interacting with the equivalent motif from the other subunit and with the sixth helix, which elongates from the catalytic domain (Figs. 1 and 2). A metal ion bound to both His42 (at 2.01 and 2.12 Å) from helices H2 of both subunits and to a chloride ion (at 2.57 Å) is shown in Fig. 1. The protein metal content analysis (see "Experimental Procedures") and the trigonal coordination geometry observed suggest that such metal ion is copper in an oxidation state I. Its location suggests a possible structural role in stabilization of the enzyme quaternary assembly for such metal ion.

Fig. 2 shows the three-dimensional structural least squares superposition of a single subunit of Ns 1,2-HQD and Ac 1,2-CTD. The first N-terminal {alpha}-helix H1 and the random coil region preceding the {alpha}-helix H4 extending from the central domain are longer in Ns 1,2-HQD compared with Ac 1,2-CTD. About one-half of the fifth helix (H5) is missing compared with the corresponding one from Ac 1,2-CTD. The first two short {beta}-sheets in Ac 1,2-CTD are also missing in Ns 1,2-HQD, being substituted by random coil regions. The secondary structure of the central section of Ns 1,2-HQD thoroughly resembles that of the 1,2-CTD family. Finally, the C-terminal region of Ns 1,2-HQD, as observed also in the Rho 1,2-CCD, the only representative of chlorocatechol cleaving dioxygenases for which the three-dimensional structure is known, lacks the seventh helix, the last long random coil, and the final {beta}-sheet present in Ac 1,2-CTD (42).



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  		FIG. 2.
Least squares superimposition of a single subunit of Ac 1,2-CTD (dark) and Ns 1,2-HQD (light) in two different orientations (A and B). C, schematic representation of the secondary structure of Ns 1,2-HQD as assigned by the program DSSP (60).

 



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  		FIG. 3.
A and B, representations of the two active site entrances of Ns 1,2-HQD. The surface is color-coded on the basis of the calculated electrostatic potential. The blue color corresponds to positive and red to negative potentials. The benzoate-like molecule is depicted in yellow. A and B show 90° vertically rotated views of the enzyme, and the two active site entrances are indicated by yellow arrows. C, Fo – Fc electron density map for the active site of Ns 1,2-HQD. The electron density is contoured at the 3{sigma} level. D, stereo representation of the superposed active sites structures of HQ docked to Ns 1,2-HQD and of the catechol complex of Ac 1,2-CTD (orange colored, PDB code 1DLT [PDB] ). Designations of amino acids positions are first given for Ns 1,2-HQD and second (in parentheses) for Ac 1,2-CTD.

 
The Catalytic Domain—Each subunit contains one catalytic pocket accessible to the substrate from the hollow side of the dimer. The central part of the catalytic domain is made up of several {beta}-sheets arranged in a {beta}-sandwich conformation and by a number of random coils positioned between the linker domain and the {beta}-sheets assembly (see Figs. 1 and 2). The active site metal center is located in the random coils region flanked on one side by the {beta}-sandwich motif of each monomer and on the other side by the {alpha}-helices of the linker domain.



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  		SCHEME 2
 
Each catalytic pocket of Ns 1,2-HQD is bordered by Leu80 and Asp83 from helix H4; Val107, Phe108, Pro110, and Phe111 from a first random coil; Trp156 from sheet S3; Tyr164, the mobile iron ligand, from a second random coil; Tyr197 from sheet S6; Ile199 and Pro200 from a third random coil; Arg218 from sheet S7; His221 and His223 from sheet S8; His237 from sheet S9; and Val251 from a fourth random coil.

As shown in Fig. 3, A and B, a distinctive feature of the present structure is that each active site presents two openings. The first one located as the one observed previously in the Ac 1,2-CTD and Rho 1,2-CCD structures and delimited by residues Leu80, Pro110, Phe111, Pro198, Ile199, Pro200 plus the backbones of the Tyr164 and Tyr197 iron ligands; the second placed at about 90° on the right side of the first one and bordered by residues Leu80, Asp83, Val107, Phe108, Gly109, and Val251. A number of water molecules are present in the openings and in the upper part of the active cavity even though a benzoate-like molecule, bound to the active site iron, occupies a large part of the cavity.

Some of the active site residues, with the corresponding Fo – Fc density overlaid are depicted in Fig. 3C. The mononuclear Fe(III) ion shows a His2Tyr2 coordination (Tyr164, Tyr197, His221, and His223), typical of all intradiol ring cleaving dioxygenases (28). X-ray absorption spectroscopy data collected for the same Ns 1,2-HQD indicate that generally the native enzyme is pentacoordinated with two spheres of atoms: either two at 1.90 Å and three at 2.06 Å, or three at 1.92 Å and two at 2.08 Å (33). In the present crystal structure, a benzoate-like ion is coordinated to the iron ion in a bidentate asymmetric mode substituting the metal bound water molecule/hydroxide ion, observed in the native 1,2-CTDs, increasing the iron coordination number to 6 (Fig. 3C). An equivalent molecule has also been observed recently in the active site of Rho 1,2-CCD. In Ns 1,2-HQD the benzoate ion is stabilized by a hydrogen bond network that connects the benzoate O1 atom to Arg218 NH1 (hydrogen-bonded further to Asp249) and the benzoate O2 atom to a well ordered W16 active site water molecule (B factor = 20.63) (hydrogen-bonded further to Phe108, Pro110, Phe111, and Trp156). As observed in Rho 1,2-CCD the benzoate binding does not trigger the dissociation of Tyr197, although causing a conformational orientation of Arg218 observed when substrates bind to intradiol dioxygenases, but contrarily to what observed in all 1,2-CTDs, 1,2-CCDs and 3,4-PCDs, Arg218, supposed to promote the substrate positioning and deprotonation, is not stabilized by a strong hydrogen bond to a Gln because this residue is replaced by His237, which is positioned a bit further away (35, 37, 38, 40, 41).

No convincing hypotheses can be made, at the moment, on the possible reasons for the presence of a benzoate-like molecule bound to the catalytic metal ion, although exogenous ligands have been often found bound to metal sites acting as stabilizers of the active enzyme by hampering metal ion dissociation. The molecule resembles benzoate or benzamide, which actually act as very weak competitive inhibitors for these enzymes, easily displaced by catechols or HQs.2

In Table II the distances of the iron ligands are reported: Tyr164 and Tyr197 exhibit shorter bonds (1.95 and 2.01 Å, respectively) than His221 and His223 (2.11 and 2.24 Å, respectively), and the iron coordination sphere is completed by the benzoate-like molecule asymmetrically bound with O1 (~2.13 Å) and O2 (~2.59 Å).


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  		TABLE II
Bond distances and angles of the catalytic iron ion coordination polyhedron for Ns 1,2-HQD (compared with Ac 1,2-CTD)

See Ref. 41.

 
Substrate Specificity—The catalytic mechanism of intradiol ring-cleaving dioxygenases has been proposed to operate via Fe(III) activation of the diol substrate first generating an Fe(II) semiquinone, which reacts directly with dioxygen to give a hydroperoxide intermediate, a Criegee rearrangement via acyl migration would finally generate the corresponding muconic anhydride (Scheme 2) (68). An alternative mechanism for migration of the electron-deficient acyl group, via a benzeneoxide-oxepin interconversion, has also been proposed (Scheme 2) (69).

Substrate selection and conversion are expected to be controlled mainly by the ring substituents effects on the electron density of the carbon atoms exposed to the molecular oxygen attack as well as by the interactions of ring substituents with the surrounding active site amino acidic residues.

Although a number of studies on inhibitors, substrates, and substrate analog adducts of 3,4-PCDs and Ac 1,2-CTD have revealed several important features of the mechanism of exogenous ligands binding to their active site, and the structure of Rho 1,2-CCD has shed some light on the substrate selectivity of chlorocatechol-cleaving enzymes, no conclusive rationalization of the observed substrate specificities for intradiol-cleaving dioxygenases has been achieved so far.

1,2-HQDs structurally belong to the group of intradiol dioxygenases comprising 1,2-CTDs. 1,2-CTDs are generally divided into two types, I and II (70). Type I dioxygenases (1,2-CTDs) are relatively specific enzymes that primarily have catechol and often also a methylcatechol as substrate. Chlorinated catechols are not used or are used only at negligible rates. Type II enzymes (better known as 1,2-CCDs) are relatively nonspecific with a wider substrate range being able to convert chlorinated catechols more rapidly than catechol and to additionally accommodate a wide range of methyl- or methoxy-substituted catechols. Regarding substrate specificity, 1,2-HQDs seem to be more closely related to type I than to the type II enzymes (Table III). Unfortunately, HQ was not tested as a potential substrate for most of the catechol and chlorocatechol 1,2-dioxygenases; a catechol 1,2-dioxygenase able to oxidize HQ at about half the rate (51%) of catechol was described only for T. cutaneum (71).


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  		TABLE III
Comparison of the substrate specificity of HQ-cleaving enzymes and two representatives of the (chloro-)catechol-cleaving enzymes

 
The Ns 1,2-HQD, subject of the present study, is remarkably substrate-selective. The conversions of HQ (Km = 1.2 µM, Vmax = 55 units/mg, kcat = 29 s–1, and kcat/Km = 24.2 µM–1 s–1), 5CHQ (2.4% relative to HQ activity), and 6CHQ (5.0% relative to HQ activity) are catalyzed (30). A range of catechols and variously substituted quinols were also tested as substrates but found to only act as inhibitors for HQ turnover (Ki ~ 10 mM for catechols).

Table III presents the comparison of the known substrate specificity data for 1,2-HQDs and some representatives of type I and II catechol-cleaving enzymes. The 1,2-HQDs from B. cepacia AC1100, Azotobacter sp. strain GP1, and T. cutaneum as well as the 1,2-CHQD from S. rochei 303, with respect to catechol, 3-methylcatechol, or pyrogallol conversion, show relatively high substrate specificities as the enzyme from N. simplex. HQ is the main substrate for the first four enzymes, whereas 6-CHQD is the best substrate for the S. rochei dioxygenase. 6-CHQD was also a relatively good substrate for the 1,2-HQDs of Azotobacter sp. GP1, N. simplex 3E, Wautersia eutropha JMP134, R. pickettii DTP0602, but was not tested with the fungal enzymes from T. cutaneum and P. chrysosporium. The enzyme from P. chrysosporium was found unable to convert 5-CHQ, but accepted catechol with 20% of the activity shown toward HQ. Also, the 1,2-HQD isolated from R. pickettii DTP0602 presents relatively low substrate specificity, being able to catalyze the oxidation of 3-methylcatechol and pyrogallol in addition to HQ and 6-CHQ, but it is inactive toward catechol, 3- and 4-chlorocatechol, 4-methylcatechol, protocatechuate, and 2,3-dihydroxybiphenyl (31). Furthermore, the 1,2-HQD of S. wittichii RW1 showed a high activity with catechol (7), and that of Arthrobacter sp. strain BA-5-17 was shown to catalyze both the intradiol and extradiol cleavage of catechol, although the activity toward HQ was 6.8-fold higher than that toward catechol (32).

These results evidence that substrate selectivity is a very heterogeneous issue even inside the 1,2-HQD group. It appears challenging to attempt the rationalization, at the molecular level, of the structural factors responsible for the differential substrate selectivity.

The structural alignment of the active site residues of the catecholate complex of Ac 1,2-CTD and the HQ docked in Ns 1,2-HQD is shown in Fig. 3D. The main interactions of Ac 1,2-CTD with the substrate involve the following residues (respective positions in the alignment of Fig. 4 given in parentheses): Leu73 (87), Pro76 (90), Ile105 (119), Pro108 (122), Leu109 (123), Arg221 (239), Phe253 (272), and Ala254 (273). Some of these residues and some additional ones in the cavity appear to be crucial in the correct positioning of the aromatic substrate in Ns 1,2-HQD (respective positions in the alignment of Fig. 4 given in parentheses): Leu80 (87), Asp83 (90), Val107 (119), Phe108 (120), Gly109 (121), Pro110 (122), Phe111 (123), Ile199 (219), Pro200 (220), Arg218 (239), and Val251 (273). We noticed substantial changes in some of these residues with respect to the corresponding amino acids in the representative structures of 1,2-CTDs and 1,2-CCDs and specifically: Asp83 (position 90 in Fig. 4, Pro76 in Ac 1,2-CTD and Ala53 in Rho 1,2-CCD), Val107 (position 119 in Fig. 4, Ile105 in Ac 1,2-CTD and Ile74 in Rho 1,2-CCD), Phe108 (position 120 in Fig. 4, Glu106 in Ac 1,2-CTD and Gln75 in Rho 1,2-CCD), Phe111 (position 123 in Fig. 4, Leu109 in Ac 1,2-CTD and Phe78 in Rho 1,2-CCD), His237 (position 258 in Fig. 4, Gln240 in Ac 1,2-CTD and Gln210 in Rho 1,2-CCD), Val251 (position 273 in Fig. 4, Ala254 in Ac 1,2-CTD and Cys224 in Rho 1,2-CCD).

To understand which of these residues could be mainly responsible for substrate recognition, HQ was docked into the active site of Ns 1,2-HQD. Two different orientations are likely: if the 4-OH substituent is oriented toward the internal part of the cavity (Fig. 3D) it would settle into a pocket formed by Asp83 and Val251 (positions 90 and 273, respectively, in Fig. 4), but if the substituent is oriented outward, it would essentially interact with Leu80 and Pro110 (positions 87 and 122 in Fig. 4, Leu73/Leu49 and Pro108/Pro77 in Ac in 1,2-CTD/Rho 1,2-CCD, respectively). This second hypothesis seems to be unlikely because identical amino acids would interact with the substrate in the different enzymes, thus not clearly accounting for their markedly different substrate specificity. Furthermore, the orientation of HQ with the 4-OH substituent toward the internal part of the cavity is equivalent to that of bound protocatechuate in 3,4-PCDs and of bound 4-methylcatechol in Ac 1,2-CTD (40, 41).



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  		FIG. 4.
Amino acid sequence alignment of several 1,2-HQDs and representatives of catechol 1,2- and chlorocatechol 1,2-dioxygenases. 1,2-HQD: N. simplex 3E (present enzyme, GenBank accession number AY822041 [GenBank] ), Arthrobacter sp. BA-5-17 (protein accession BAA82713 [GenBank] , R. opacus SAO101 (protein accession BAD30043 [GenBank] , R. pickettii DTP0602 (protein accession BAA13107 [GenBank] , S. wittichii RW1 (protein accession CAA51371 [GenBank] , B. cepacia AC1100 (protein accession AAC43338 [GenBank] , A. tumefaciens strain C58 (protein accession AAK88258 [GenBank] ; W. eutropha JMP134 (protein accession AAM55216 [GenBank] , 1,2-CTD: Arthrobacter sp. mA3 (protein accession JT0613), R. opacus 1CP (protein accession CAA67941 [GenBank] , Acinetobacter sp. ADP1 (protein accession YP_046127), Pseudomonas putida PRS2000 (protein accession AAA66204 [GenBank] ; 1,2-CCD: R. opacus 1CP (p1CP) (protein accession AAC38251 [GenBank] , R. opacus 1CP (p1CP) (protein accession CAD28142 [GenBank] , W. eutropha JMP134 (pJP4) (protein accession AAA98262 [GenBank] , P. putida AC866 (pAC27) (protein accession CAE92861 [GenBank] . Alignment positions identical in all sequences are marked with a black background. Regions in which the sequences of all 1,2-HQDs are identical but differ from those of catechol or chlorocatechol 1,2-dioxygenases, or the other way around, are marked with light gray. Single amino acid positions marked dark gray are important for substrate specificity and discussed in the text.

 
A multiple sequence alignment of intradiol dioxygenases revealed several positions identical in all available 1,2-HQDs but different in one or more of the selected 1,2-CTDs or 1,2-CCDs (shaded dark gray in Fig. 4). Such positions may be conserved without a specific selection pressure, but they also may be of importance for the substrate specificity without a structural reason for it so far having been elucidated.

The presence of Asp83 and Val251 (positions 90 and 273 in Fig. 4), the residues expected to interact with substituents in position 4 (Pro76/Ala53 and Ala254/Cys224 in Ac 1,2-CTD/Rho 1,2CCD, respectively), based on structural comparisons should be mainly responsible for the selective preference for HQs. Valine residues corresponding to Val251 (position 273 in Fig. 4) occur in all available 1,2-HQDs, but not in the (chloro-)catechol dioxygenases, a distribution supporting the conclusions drawn from structural comparisons.

Also, 3,4-PCDs are selective for a substrate carrying a hydrophilic substituent in a distal position with respect to the diol: protocatechuate. It has been shown that the carboxyl group of protocatechuate forms a hydrogen bond with Tyr324 (substituted by Asp83 in 1,2-HQD), and its negative charge is complemented by long range electrostatic interactions with Arg133, Arg330, and Arg450 (Ac 3,4-PCD numbering) (40). In Ns 1,2-HQD Asp83 could have a function similar to that of Tyr324 in 3,4-PCDs, although in the case of HQ the substrate should not be charged at neutral pH but, Asp83 would.

The other feature crucial for HQ selection and limited affinity for catechols is the presence of the second large active site opening caused by the substitution and spatial reallocation of residues Gly72 and Asp81 in Ac 1,2-CTD with Ile79 and Ser88, amino acids specifically conserved in all 1,2-HQDs (positions 86 and 100, respectively in Fig. 4). Contributing to the second large active site opening is a marked shift of Pro110 (position 122 in Fig. 4, Pro108 in Ac 1,2-CTD). The resulting larger solvent exposition of the upper part of the cavity should electrostatically favor HQ settling and stabilization.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
The crystal structure of Ns 1,2-HQD evidences significant differences with respect to the known 1,2-CCD and 1,2-CTD enzymes. Several secondary structure differences are noticed, but more importantly a number of residues within the active cleft and believed to be responsible for substrate selection are altered. Leu80, Asp83, Val107, Phe108, Gly109, Pro110, Phe111, Ile199, Pro200, Arg218, and Val251 seem to be directly involved in interactions with substrates. Few amino acid differences (Asp83 and Val251) between the N. simplex enzyme which exhibits a marked selectivity toward HQ, 1,2-CCDs (Ala53 and Cys224, respectively, in Rho 1,2CCD) and 1,2-CTDs (Pro76 and Ala254, respectively, in Ac 1,2-CTD) as well as the extensive solvent exposure of the upper part of the catalytic site are believed to be responsible for the observed substrate selectivity differences among 1,2-HQDs and the other intradiol ring-cleaving dioxygenases.


   FOOTNOTES
 
* This work was supported by Grant ICA2-CT-2000-10006 from the European Commission Research and Technological Development Program Copernicus, Contract HPRI-CT-1999-00017 from the European Community Access to Research Infrastructure Action of the Improving Human Potential Program to the EMBL Hamburg outstation, and Grant COFIN2002 from the Italian Ministero Università e Ricerca Scientifica. 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.

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

|| To whom correspondence should be addressed. Fax: 39-055-457-3333; E-mail: fabrizio.briganti{at}unifi.it.

1 The abbreviations used are: HQ, hydroxyquinol; 1,2-CCD, chlorocatechol 1,2-dioxygenase; 5CHQ, 5-chlorohydroxyquinol; 6CHQ, 6-chlorohydroxyquinol; 1,2-CHQD, chlorohydroxyquinol 1,2-dioxygenase; 1,2-CTD, catechol 1,2-dioxygenase; 1,2-HQD, hydroxyquinol 1,2-dioxygenase; MAD, multiple wavelength anomalous dispersion; 3,4-PCD, protocatechuate 3,4-dioxygenase. Back

2 M. Ferraroni, A. Scozzafava, and F. Briganti, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank V. Nödinger and R. Schmid, Universität Stuttgart, for N-terminal and peptide sequencing.



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