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• W2058325166 abstract "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. 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. Hydroxyquinol (1,2,4-trihydroxybenzene) (HQ) 1The 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. 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 (1Sze I.S. Dagley S. J. Bacteriol. 1984; 159: 353-359Crossref PubMed Google Scholar, 2Rieble S. Joshi D.K. Gold M.H. J. Bacteriol. 1994; 176: 4838-4844Crossref PubMed Google Scholar, 3Anderson J.J. Dagley S. J. Bacteriol. 1980; 141: 534-543Crossref PubMed Google Scholar). In bacteria HQ has been identified as an intermediate in the degradation of mononuclear hydroxyaromatic compounds such as resorcinol and 2,4-dihydroxybenzoate (4Chapman P.J. Ribbons D.W. J. Bacteriol. 1976; 125: 975-984Crossref PubMed Google Scholar, 5Stolz A. Knackmuss H.J. FEMS Microbiol. Lett. 1993; 108: 219-224Crossref PubMed Scopus (0) Google Scholar) or amino-hydroxyaromatic compounds such as 4-aminophenol (6Takenaka S. Okugawa S. Kadowaki M. Murakami S. Aoki K. Appl. Environ. Microbiol. 2003; 69: 5410-5413Crossref PubMed Scopus (62) Google Scholar) as well as in the degradation of hydroxylated biaryl ethers such as 2-hydroxydibenzo-p-dioxin and 3-hydroxydibenzofuran (7Armengaud J. Timmis K.N. Wittich R.M. J. Bacteriol. 1999; 181: 3452-3461Crossref PubMed Google Scholar). HQ also occurs in the catabolic pathways of aromatic compounds carrying nitro groups such as in 3- and 4-nitrophenol or as 4-nitrocatechol (8Chauhan A. Samanta S.K. Jain R.K. J. Appl. Microbiol. 2000; 88: 764-772Crossref PubMed Scopus (42) Google Scholar, 9Chauhan A. Chakraborti A.K. Jain R.K. Biochem. Biophys. Res. Commun. 2000; 270: 733-740Crossref PubMed Scopus (77) Google Scholar, 10Jain R.K. Dreisbach J.H. Spain J.C. Appl. Environ. Microbiol. 1994; 60: 3030-3032Crossref PubMed Google Scholar, 11Kitagawa W. Kimura N. Kamagata Y. J. Bacteriol. 2004; 186: 4894-4902Crossref PubMed Scopus (152) Google Scholar, 12Meulenberg R. Pepi M. de Bont J.A. Biodegradation. 1996; 7: 303-311Crossref PubMed Scopus (48) Google Scholar). 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 (13Apajalahti J.H. Salkinoja-Salonen M.S. J. Bacteriol. 1987; 169: 5125-5130Crossref PubMed Google Scholar, 14Uotila J.S. Kitunen V.H. Coote T. Saastamoinen T. Salkinoja-Salonen M. Apajalahti J.H. Biodegradation. 1995; 6: 119-126Crossref PubMed Scopus (14) Google Scholar, 15Häggblom M.M. Janke D. Salkinoja-Salonen M. J. Bacteriol. 1989; 55: 516-519Google Scholar, 16Uotila J.S. Kitunen V.H. Saastamoinen T. Coote T. Häggblom M.M. Salkinoja-Salonen M.S. J. Bacteriol. 1992; 174: 5669-5675Crossref PubMed Google Scholar), 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 (17Xun L. Bohuslavek J. Cai M. Biochem. Biophys. Res. Commun. 1999; 266: 322-325Crossref PubMed Scopus (53) Google Scholar). 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 (18Golovleva L.A. Zaborina O. Pertsova R. Baskunov B. Schurukhin Y. Kuzmin S. Biodegradation. 1992; 2: 201-208Crossref Scopus (64) Google Scholar, 19Kiyohara H. Hatta T. Ogawa Y. Kakuda T. Yokoyama H. Takizawa N. Appl. Environ. Microbiol. 1992; 58: 1276-1283Crossref PubMed Google Scholar, 20Latus M. Seitz H.-J. Eberspächer J. Lingens F. Appl. Environ. Microbiol. 1995; 61: 2453-2460Crossref PubMed Google Scholar, 21Louie T.M. Webster C.M. Xun L. J. Bacteriol. 2002; 184: 3492-3500Crossref PubMed Scopus (111) Google Scholar, 22Padilla L. Matus V. Zenteno P. Gonzalez B. J. Basic Microbiol. 2000; 40: 243-249Crossref PubMed Scopus (35) Google Scholar, 23Xun L. Webster C.M. J. Biol. Chem. 2004; 279: 6696-6700Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 24Zaborina O. Latus M. Eberspächer J. Golovleva L.A. Lingens F. J. Bacteriol. 1995; 177: 229-234Crossref PubMed Google Scholar). 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 (18Golovleva L.A. Zaborina O. Pertsova R. Baskunov B. Schurukhin Y. Kuzmin S. Biodegradation. 1992; 2: 201-208Crossref Scopus (64) Google Scholar, 19Kiyohara H. Hatta T. Ogawa Y. Kakuda T. Yokoyama H. Takizawa N. Appl. Environ. Microbiol. 1992; 58: 1276-1283Crossref PubMed Google Scholar, 24Zaborina O. Latus M. Eberspächer J. Golovleva L.A. Lingens F. J. Bacteriol. 1995; 177: 229-234Crossref PubMed Google Scholar). In 2,4,5-trichlorophenoxyacetate degradation by Burkolderia (Pseudomonas) cepacia AC1100 5CHQ and HQ are formed sequentially as intermediates (25Daubaras D.L. Saido K. Chakrabarty A.M. Appl. Environ. Microbiol. 1996; 62: 4276-4279Crossref PubMed Google Scholar). 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 (26Kozyreva L.P. Shurukhin I. Finkelshtein Z.I. Baskunov B. Golovleva L.A. Microbiology (translation of Mikrobiologiya). 1993; 62: 78-85Google Scholar, 27Solyanikova I.P. Protopopova Y.Y. Travkin V. Golovleva L.A. Biochemistry (Moscow). 1996; 61: 461-466Google Scholar). 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 (28Lange S.J. Que Jr., L. Curr. Opin. Chem. Biol. 1998; 2: 159-172Crossref PubMed Scopus (167) Google Scholar). 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) (29Pieper D.H. Pollmann K. Nikodem P. Gonzalez B. Wray V. J. Bacteriol. 2002; 184: 1466-1470Crossref PubMed Scopus (22) Google Scholar). 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 (1Sze I.S. Dagley S. J. Bacteriol. 1984; 159: 353-359Crossref PubMed Google Scholar, 2Rieble S. Joshi D.K. Gold M.H. J. Bacteriol. 1994; 176: 4838-4844Crossref PubMed Google Scholar, 6Takenaka S. Okugawa S. Kadowaki M. Murakami S. Aoki K. Appl. Environ. Microbiol. 2003; 69: 5410-5413Crossref PubMed Scopus (62) Google Scholar, 20Latus M. Seitz H.-J. Eberspächer J. Lingens F. Appl. Environ. Microbiol. 1995; 61: 2453-2460Crossref PubMed Google Scholar, 24Zaborina O. Latus M. Eberspächer J. Golovleva L.A. Lingens F. J. Bacteriol. 1995; 177: 229-234Crossref PubMed Google Scholar, 25Daubaras D.L. Saido K. Chakrabarty A.M. Appl. Environ. Microbiol. 1996; 62: 4276-4279Crossref PubMed Google Scholar, 30Travkin V.M. Jadan A.P. Briganti F. Scozzafava A. Golovleva L.A. FEBS Lett. 1997; 407: 69-72Crossref PubMed Scopus (29) Google Scholar, 31Hatta T. Nakano O. Imai N. Takizawa N. Kiyohara H. J. Biosci. Bioeng. 1999; 87: 267-272Crossref PubMed Scopus (36) Google Scholar, 32Murakami S. Okuno T. Matsumura E. Takenaka S. Shinke R. Aoki K. Biosci. Biotechnol. Biochem. 1999; 63: 859-865Crossref PubMed Scopus (21) Google Scholar). 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 (α Fe(III))2 (30Travkin V.M. Jadan A.P. Briganti F. Scozzafava A. Golovleva L.A. FEBS Lett. 1997; 407: 69-72Crossref PubMed Scopus (29) Google Scholar). 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 (2Rieble S. Joshi D.K. Gold M.H. J. Bacteriol. 1994; 176: 4838-4844Crossref PubMed Google Scholar, 33Briganti F. Mangani S. Pedocchi L. Scozzafava A. Golovleva L.A. Jadan A.P. Solyanikova I.P. FEBS Lett. 1998; 433: 58-62Crossref PubMed Scopus (22) Google Scholar). 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 (34Ohlendorf D.H. Lipscomb J.D. Weber P.C. Nature. 1988; 336: 403-405Crossref PubMed Scopus (250) Google Scholar, 35Elgren T.E. Orville A.M. Kelly K.A. Lipscomb J.D. Ohlendorf D.H. Que Jr., L. Biochemistry. 1997; 36: 11504-11513Crossref PubMed Scopus (77) Google Scholar, 36Ohlendorf D.H. Orville A.M. Lipscomb J.D. J. Mol. Biol. 1994; 244: 586-608Crossref PubMed Scopus (166) Google Scholar, 37Orville A.M. Elango N. Lipscomb J.D. Ohlendorf D.H. Biochemistry. 1997; 36: 10039-10051Crossref PubMed Scopus (77) Google Scholar, 38Orville A.M. Lipscomb J.D. Ohlendorf D.H. Biochemistry. 1997; 36: 10052-10066Crossref PubMed Scopus (165) Google Scholar, 39D'Argenio D.A. Vetting M.W. Ohlendorf D.H. Ornston L.N. J. Bacteriol. 1999; 181: 6478-6487Crossref PubMed Google Scholar, 40Vetting M.W. D'Argenio D.A. Ornston L.N. Ohlendorf D.H. Biochemistry. 2000; 39: 7943-7955Crossref PubMed Scopus (94) Google Scholar). 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 (41Vetting M.W. Ohlendorf D.H. Struct. Fold. Des. 2000; 8: 429-440Abstract Full Text Full Text PDF Scopus (123) Google Scholar, 42Ferraroni M. Solyanikova I.P. Kolomytseva M.P. Scozzafava A. Golovleva L.A. Briganti F. J. Biol. Chem. 2004; 279: 27646-27655Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). DNA sequencing showed that 1,2-HQDs are most closely related to catechol and chlorocatechol dioxygenases (7Armengaud J. Timmis K.N. Wittich R.M. J. Bacteriol. 1999; 181: 3452-3461Crossref PubMed Google Scholar, 11Kitagawa W. Kimura N. Kamagata Y. J. Bacteriol. 2004; 186: 4894-4902Crossref PubMed Scopus (152) Google Scholar, 21Louie T.M. Webster C.M. Xun L. J. Bacteriol. 2002; 184: 3492-3500Crossref PubMed Scopus (111) Google Scholar, 31Hatta T. Nakano O. Imai N. Takizawa N. Kiyohara H. J. Biosci. Bioeng. 1999; 87: 267-272Crossref PubMed Scopus (36) Google Scholar, 32Murakami S. Okuno T. Matsumura E. Takenaka S. Shinke R. Aoki K. Biosci. Biotechnol. Biochem. 1999; 63: 859-865Crossref PubMed Scopus (21) Google Scholar, 43Daubaras D.L. Hershberger C.D. Kitano K. Chakrabarty A.M. Appl. Environ. Microbiol. 1995; 61: 1279-1289Crossref PubMed Google Scholar). Nevertheless, 1,2-HQDs appear to have a distinct substrate specificity and do not, or relatively slowly, convert catechol or substituted catechols, respectively (1Sze I.S. Dagley S. J. Bacteriol. 1984; 159: 353-359Crossref PubMed Google Scholar, 2Rieble S. Joshi D.K. Gold M.H. J. Bacteriol. 1994; 176: 4838-4844Crossref PubMed Google Scholar, 6Takenaka S. Okugawa S. Kadowaki M. Murakami S. Aoki K. Appl. Environ. Microbiol. 2003; 69: 5410-5413Crossref PubMed Scopus (62) Google Scholar, 7Armengaud J. Timmis K.N. Wittich R.M. J. Bacteriol. 1999; 181: 3452-3461Crossref PubMed Google Scholar, 20Latus M. Seitz H.-J. Eberspächer J. Lingens F. Appl. Environ. Microbiol. 1995; 61: 2453-2460Crossref PubMed Google Scholar, 22Padilla L. Matus V. Zenteno P. Gonzalez B. J. Basic Microbiol. 2000; 40: 243-249Crossref PubMed Scopus (35) Google Scholar, 24Zaborina O. Latus M. Eberspächer J. Golovleva L.A. Lingens F. J. Bacteriol. 1995; 177: 229-234Crossref PubMed Google Scholar, 25Daubaras D.L. Saido K. Chakrabarty A.M. Appl. Environ. Microbiol. 1996; 62: 4276-4279Crossref PubMed Google Scholar, 30Travkin V.M. Jadan A.P. Briganti F. Scozzafava A. Golovleva L.A. FEBS Lett. 1997; 407: 69-72Crossref PubMed Scopus (29) Google Scholar, 31Hatta T. Nakano O. Imai N. Takizawa N. Kiyohara H. J. Biosci. Bioeng. 1999; 87: 267-272Crossref PubMed Scopus (36) Google Scholar, 32Murakami S. Okuno T. Matsumura E. Takenaka S. Shinke R. Aoki K. Biosci. Biotechnol. Biochem. 1999; 63: 859-865Crossref PubMed Scopus (21) Google Scholar). 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 (30Travkin V.M. Jadan A.P. Briganti F. Scozzafava A. Golovleva L.A. FEBS Lett. 1997; 407: 69-72Crossref PubMed Scopus (29) Google Scholar), 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. 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 (44Golovleva L.A. Pertsova R.N. Evtushenko L.I. Baskunov B.P. Biodegradation. 1990; 1: 263-271Crossref PubMed Scopus (32) Google Scholar). 1,2-HQD from N. simplex 3E was purified as reported previously (30Travkin V.M. Jadan A.P. Briganti F. Scozzafava A. Golovleva L.A. FEBS Lett. 1997; 407: 69-72Crossref PubMed Scopus (29) Google Scholar). Tryptic peptides were isolated as described for other enzymes (45Eulberg D. Golovleva L.A. Schlömann M. J. Bacteriol. 1997; 179: 370-381Crossref PubMed Google Scholar, 46Stone K.L. LoPresti M.B. Crawford J.M. DeAngelis R. Williams K.R. Matsudaira P.T. A Practical Guide to Protein and Peptide Purification for Microsequencing. Academic Press, San Diego1989: 33-47Google Scholar) 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 (47Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar). pBluescript II SK(+) obtained from Stratagene was used as general cloning vector, and a T vector (48Marchuk D. Drumm M. Saulino A. Collins F.S. Nucleic Acids Res. 1991; 19: 1154Crossref PubMed Scopus (1130) Google Scholar) derived from it was used for cloning PCR products. Recombinant plasmids were transformed into E. coli DH5α, bought from Invitrogen. Genomic DNA from N. simplex 3E was prepared by the method of Wilson (49Wilson K. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Struhl K. Current Protocols in Molecular Biology. 1. John Wiley & Sons, Inc., New York2000: 2.4.1-2.4.5Google Scholar). 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 (7Armengaud J. Timmis K.N. Wittich R.M. J. Bacteriol. 1999; 181: 3452-3461Crossref PubMed Google Scholar, 31Hatta T. Nakano O. Imai N. Takizawa N. Kiyohara H. J. Biosci. Bioeng. 1999; 87: 267-272Crossref PubMed Scopus (36) Google Scholar, 32Murakami S. Okuno T. Matsumura E. Takenaka S. Shinke R. Aoki K. Biosci. Biotechnol. Biochem. 1999; 63: 859-865Crossref PubMed Scopus (21) Google Scholar, 43Daubaras D.L. Hershberger C.D. Kitano K. Chakrabarty A.M. Appl. Environ. Microbiol. 1995; 61: 1279-1289Crossref PubMed Google Scholar). 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 × 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 (47Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar). 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 DH5α, 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. Comparisons with data-base entries were performed by using BLASTX (50Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar). Multiple sequence alignments were created using ClustalX (version 1.8) (51Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35620) Google Scholar). 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 (52Benvenuti M. Briganti F. Scozzafava A. Golovleva L.A. Travkin V.M. Mangani S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 901-903Crossref PubMed Scopus (9) Google Scholar). 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, β = 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 (53Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). The program SOLVE (54Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar) 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 (55Collaborative Computational P.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). Automatic tracing was performed initially with the program RESOLVE (56Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1755-1762Crossref PubMed Scopus (166) Google Scholar) and extended using ARP/WARP version 6.0 (57Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2565) Google Scholar). 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 (55Collaborative Computational P.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). Manual rebuilding of the model was performed using the program QUANTA (58Quanta v. 98 Quanta Simulation, Search and Analysis. Molecular Simulation, Inc., San Diego1997Google Scholar). Solvent molecules were introduced automatically using ARP (57Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2565) Google Scholar). 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.Table ISummary of data collection and atomic model refinement statisticsData collectionNativeFe peakFe inflectionFe remoteWavelength (Å)0.9081.7381.7411.001Limiting resolution (Å)1.752.62.62.2Unique reflections61,45418,84118,76835,271Rsym(%)4.6 (11.0)bNumbers in parentheses are for the highest resolution shell.6.4 (21.4)6.2 (19.1)4.7 (31.0)Multiplicity4.35.13.23.7Completeness overall (%)94.1 (69.6)99.5 (95.7)99.1 (91.8)99.8 (98.3)〈I / σ(I)〉22.2 (6.4)18.4 (4.2)14.4 (3.4)23.3 (3.6)Overall figure of meritBefore density modification0.56After density modification0.82RefinementResolution range (Å)20.0-1.75Unique reflections, working/free56,787/2,988R factor (%)19.2Rfree (%)24.6No. of non-hydrogen atoms4,541No. of water molecules837r.m.s.d.ar.m.s.d., root mean square difference. bond length (Å)0.017r.m.s.d. bond angle (°)1.588a r.m.s.d., root mean square difference.b Numbers in parentheses are for the highest resolution shell. Open table in a new tab Protein coordinates have been deposited in the Protein Data Bank (accession number 1TMX). 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 12σ 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 b" @default.
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• W2058325166 date "2005-06-01" @default.
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• W2058325166 title "Crystal Structure of the Hydroxyquinol 1,2-Dioxygenase from Nocardioides simplex 3E, a Key Enzyme Involved in Polychlorinated Aromatics Biodegradation" @default.
• W2058325166 cites W1498414175 @default.
• W2058325166 cites W1520730837 @default.
• W2058325166 cites W1524642401 @default.
• W2058325166 cites W1539796472 @default.
• W2058325166 cites W1574688274 @default.
• W2058325166 cites W1584487973 @default.
• W2058325166 cites W1591191945 @default.
• W2058325166 cites W1632346421 @default.
• W2058325166 cites W1675690491 @default.
• W2058325166 cites W169362524 @default.
• W2058325166 cites W1776261964 @default.
• W2058325166 cites W1877355868 @default.
• W2058325166 cites W1877788388 @default.
• W2058325166 cites W1919603473 @default.
• W2058325166 cites W1933540506 @default.
• W2058325166 cites W1965277349 @default.
• W2058325166 cites W1968059064 @default.
• W2058325166 cites W1970018993 @default.
• W2058325166 cites W1971439087 @default.
• W2058325166 cites W1977471578 @default.
• W2058325166 cites W1981689753 @default.
• W2058325166 cites W1986191025 @default.
• W2058325166 cites W1997100196 @default.
• W2058325166 cites W1997425794 @default.
• W2058325166 cites W1999312426 @default.
• W2058325166 cites W2001641653 @default.
• W2058325166 cites W2006946556 @default.
• W2058325166 cites W2008708467 @default.
• W2058325166 cites W2009108675 @default.
• W2058325166 cites W2010984666 @default.
• W2058325166 cites W2014098870 @default.
• W2058325166 cites W2014126369 @default.
• W2058325166 cites W2018949333 @default.
• W2058325166 cites W2029508736 @default.
• W2058325166 cites W2032091243 @default.
• W2058325166 cites W2033613795 @default.
• W2058325166 cites W2041456458 @default.
• W2058325166 cites W2044179958 @default.
• W2058325166 cites W2052895859 @default.
• W2058325166 cites W2059269003 @default.
• W2058325166 cites W2064825632 @default.
• W2058325166 cites W2068254002 @default.
• W2058325166 cites W2075271223 @default.
• W2058325166 cites W2079985200 @default.
• W2058325166 cites W2087345559 @default.
• W2058325166 cites W2090640471 @default.
• W2058325166 cites W2091630069 @default.
• W2058325166 cites W2092670056 @default.
• W2058325166 cites W2097382368 @default.
• W2058325166 cites W2098204522 @default.
• W2058325166 cites W2099040998 @default.
• W2058325166 cites W2104051423 @default.
• W2058325166 cites W2105321472 @default.
• W2058325166 cites W2111095768 @default.
• W2058325166 cites W2128585170 @default.
• W2058325166 cites W2131852827 @default.
• W2058325166 cites W2139918194 @default.
• W2058325166 cites W2156666818 @default.
• W2058325166 cites W2158714788 @default.
• W2058325166 cites W2163286689 @default.
• W2058325166 cites W2172082961 @default.
• W2058325166 cites W4245017398 @default.
• W2058325166 cites W4293003482 @default.
• W2058325166 doi "https://doi.org/10.1074/jbc.m500666200" @default.
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