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Lawrence P. Wackett - One of the best experts on this subject based on the ideXlab platform.
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the role of histidine 200 in mndd the mn ii dependent 3 4 dihydroxyphenylacetate 2 3 dioxygenase from Arthrobacter Globiformis cm 2 a site directed mutagenesis study
Journal of Biological Inorganic Chemistry, 2005Co-Authors: Joseph P Emerson, Michael J. Sadowsky, Lawrence Que, M L Wagner, Mark F Reynolds, Lawrence P. WackettAbstract:The manganese-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase (MndD) from Arthrobacter Globiformis CM-2 is an extradiol-cleaving catechol dioxygenase that catalyzes aromatic ring cleavage of 3,4-dihydroxyphenylacetate (DHPA). Based on the recent crystal structure of the MndD–DHPA complex, a series of site-directed mutations were made at a conserved second-sphere residue, histidine 200, to gain insight into and clarify the role this residue plays in the Mn(II)-dependent catalytic mechanism. In this study, we report the activities and spectroscopic data of these H200 variants and their DHPA and 4-nitrocatechol (4-NC) complexes. The data collected from wild-type and mutant MndDs are consistent with a role for H200 interacting with a manganese-bound dioxygen moiety and are inconsistent with other previously proposed roles involving proton transfer. Spectroscopic observations, including unique low-field EPR signals found when DHPA and 4-NC are bound to the Mn(II) center of MndD, are discussed and their relationship to dioxygen activation catalyzed in MndD is explored.
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manganese ii active site mutants of 3 4 dihydroxyphenylacetate 2 3 dioxygenase from Arthrobacter Globiformis strain cm 2
Biochemistry, 1997Co-Authors: Yvonne R. Boldt, Michael J. Sadowsky, Lawrence Que, Adam K Whiting, M L Wagner, Lawrence P. WackettAbstract:Whereas all other members of the extradiol-cleaving catechol dioxygenase family are iron-dependent, the 3,4-dihydroxyphenylacetate 2,3-dioxygenase (MndD) from Arthrobacter Globiformis CM-2 is dependent on manganese for catalytic activity. Recently, the endogenous iron ligands of one family member, the 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC), were identified crystallographically as two histidines and a glutamic acid [Sugiyama, K., et al. (1995) Proc. Jpn. Acad., Ser. B 71, 32-35; Han, et al. (1995) Science 270, 976-980; Senda, T., et al. (1996) J. Mol. Biol. 255, 735-752]. Though BphC and MndD have low overall sequence identity (23%), the three BphC metal ligands are all conserved in MndD (H155, H214, and E266). In order to determine whether these residues also act as ligands to manganese in MndD, site-directed mutants of each were constructed, purified, and analyzed for activity and metal content. Mutations H155A, H214A, and E266Q yielded purified enzymes with specific activities of <0.1% of that of the wild-type dioxygenase and bound 0.4, 1.8, and 33% of the wild-type level of manganese, respectively. The relatively high level of manganese [with a Mn(II) EPR signal distinctly different from that of the wild-type enzyme] observed for E266Q suggests that the glutamine may act as a weak ligand to the metal. Mutant E266D, which retains the potential metal binding capability of a carboxylate group, exhibited 12% of the wild-type activity in crude extracts, suggesting that Mn remains bound; however, this mutant protein was too unstable to be purified and analyzed for metal content. On the basis of the low activity and metal content of mutant proteins, we propose that the conserved residues H155, H214, and E266 ligate manganese in MndD. As is the case with the superoxide dismutases, the extradiol-cleaving catechol dioxygenases appear to utilize identical coordinating residues for their iron- and manganese-dependent enzymes.
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manganese ii dependent extradiol cleaving catechol dioxygenase from Arthrobacter Globiformis cm 2
Biochemistry, 1996Co-Authors: Adam K Whiting, Yvonne R. Boldt, Lawrence P. Wackett, Michael P Hendrich, Lawrence QueAbstract:A manganese-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase from Arthrobacter Globiformis strain CM-2 (MndD) cloned in Escherichia coli has been purified to homogeneity. Sedimentation equilibrium analysis indicates an α4 homotetrameric holoenzyme structure (4 × 38 861 Da). Steady-state kinetic analysis of MndD with a variety of substrates and inhibitors yields very similar relative rates to the known Fe(II)- and Mn(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenases from Pseudomonas ovalis and Bacillus brevis, respectively. Yet, unlike the Fe(II)-dependent enzyme, MndD retains almost all activity in the presence of H2O2 and CN- and is inactivated by Fe(II). ICP emission analysis confirms the presence of 3.0 ± 0.2 g-atoms Mn (and only 0.7 ± 0.2 g-atoms Fe) per tetrameric holoenzyme molecule. Comparison of MndD samples with varying metal content, including an apo and partial-apo enzyme preparation, shows a strong positive correlation between specific activity and Mn content. EPR spectra of MndD a...
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A manganese-dependent dioxygenase from Arthrobacter Globiformis CM-2 belongs to the major extradiol dioxygenase family.
Journal of bacteriology, 1995Co-Authors: Yvonne R. Boldt, Michael J. Sadowsky, L. B. M. Ellis, Lawrence Que, Lawrence P. WackettAbstract:Almost all bacterial ring cleavage dioxygenases contain iron as the catalytic metal center. We report here the first available sequence for a manganese-dependent 3,4-dihydroxyphenylacetate (3,4-DHPA) 2,3-dioxygenase and its further characterization. This manganese-dependent extradiol dioxygenase from Arthrobacter Globiformis CM-2, unlike iron-dependent extradiol dioxygenases, is not inactivated by hydrogen peroxide. Also, ferrous ions, which activate iron extradiol dioxygenases, inhibit 3,4-DHPA 2,3-dioxygenase. The gene encoding 3,4-DHPA 2,3-dioxygenase, mndD, was identified from an A. Globiformis CM-2 cosmid library. mndD was subcloned as a 2.0-kb SmaI fragment in pUC18, from which manganese-dependent extradiol dioxygenase activity was expressed at high levels in Escherichia coli. The mndD open reading frame was identified by comparison with the known N-terminal amino acid sequence of purified manganese-dependent 3,4-DHPA 2,3-dioxygenase. Fourteen of 18 amino acids conserved in members of the iron-dependent extradiol dioxygenase family are also conserved in the manganese-dependent 3,4-DHPA 2,3-dioxygenase (MndD). Thus, MndD belongs to the extradiol family of dioxygenases and may share a common ancestry with the iron-dependent extradiol dioxygenases. We propose the revised consensus primary sequence (G,T,N,R)X(H,A)XXXXXXX(L,I,V,M,F)YXX(D,E,T,N,A)PX(G,P) X(2,3)E for this family. (Numbers in brackets indicate a gap of two or three residues at this point in the sequence.) The suggested common ancestry is also supported by sequence obtained from genes flanking mndD, which share significant sequence identity with xylJ and xylG from Pseudomonas putida.
David M. Dooley - One of the best experts on this subject based on the ideXlab platform.
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characterization of the preprocessed copper site equilibrium in amine oxidase and assignment of the reactive copper site in topaquinone biogenesis
Journal of the American Chemical Society, 2019Co-Authors: Charles N Adelson, Eric M. Shepard, David M. Dooley, Doreen E Brown, Kimberly M Hilmer, Esther M Johnston, Hope Watts, Joan B Broderick, Edward I SolomonAbstract:Copper-dependent amine oxidases produce their redox active cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), via the CuII-catalyzed oxygenation of an active site tyrosine. This study addresses possible mechanisms for this biogenesis process by presenting the geometric and electronic structure characterization of the CuII-bound, prebiogenesis (preprocessed) active site of the enzyme Arthrobacter Globiformis amine oxidase (AGAO). CuII-loading into the preprocessed AGAO active site is slow (kobs = 0.13 h–1), and is preceded by CuII binding in a separate kinetically favored site that is distinct from the active site. Preprocessed active site CuII is in a thermal equilibrium between two species, an entropically favored form with tyrosine protonated and unbound from the CuII site, and an enthalpically favored form with tyrosine bound deprotonated to the CuII active site. It is shown that the CuII-tyrosinate bound form is directly active in biogenesis. The electronic structure determined for the reactive fo...
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kinetics and spectroscopic evidence that the cu i semiquinone intermediate reduces molecular oxygen in the oxidative half reaction of Arthrobacter Globiformis amine oxidase
Biochemistry, 2008Co-Authors: Eric M. Shepard, Kristina M Okonski, David M. DooleyAbstract:The role of copper during the reoxidation of substrate-reduced amine oxidases by O2 has not yet been definitively established. Both outer-sphere and inner-sphere pathways for the reduction of O2 to H2O2 have been proposed. A key step in the inner-sphere mechanism is the reaction of O2 directly with the Cu(I) center of a Cu(I)−semiquinone intermediate. To thoroughly examine this possibility, we have measured the spectral changes associated with single-turnover reoxidation by O2 of substrate-reduced Arthrobacter Globiformis amine oxidase (AGAO) under a wide range of conditions. We have previously demonstrated that the internal electron-transfer reaction [Cu(II)−TPQAMQ → Cu(I)−TPQSQ] (where TPQAMQ is the aminoquinol form of reduced TPQ and TPQSQ is the semiquinone form) occurs at a rate that could permit the reaction of O2 with both species to be observed on the stopped-flow time scale [Shepard, E. M., and Dooley, D. M. (2006) J. Biol. Inorg. Chem. 11, 1039−1048]. The transient absorption spectra observed fo...
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complexes of the copper containing amine oxidase from Arthrobacter Globiformis with the inhibitors benzylhydrazine and tranylcypromine
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2008Co-Authors: David B Langley, David M. Dooley, Daniel M Trambaiolo, Anthony P Duff, H C Freeman, Mitchell J GussAbstract:Complexes of Arthrobacter Globiformis amine oxidase (AGAO) with the inhibitors benzylhydrazine and tranylcypromine (an antidepressant drug) have been refined at 1.86 and 1.65 A resolution, respectively. Both inhibitors form covalent adducts with the TPQ cofactor. A tyrosine residue, proposed to act as a gate to the AGAO active site, is in its open conformation.
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enantiomer specific binding of ruthenium ii molecular wires by the amine oxidase of Arthrobacter Globiformis
Journal of the American Chemical Society, 2008Co-Authors: David B Langley, David M. Dooley, Anthony P Duff, Mitchell J Guss, Harry B Gray, Doreen E Brown, Lionel Cheruzel, Stephen M Contakes, Kimberly M Hilmer, H C FreemanAbstract:The copper amine oxidase from Arthrobacter Globiformis (AGAO) is reversibly inhibited by molecular wires comprising a Ru(II) complex head group and an aromatic tail group joined by an alkane linker. The crystal structures of a series of Ru(II)-wire−AGAO complexes differing with respect to the length of the alkane linker have been determined. All wires lie in the AGAO active-site channel, with their aromatic tail group in contact with the trihydroxyphenylalanine quinone (TPQ) cofactor of the enzyme. The TPQ cofactor is consistently in its active (“off-Cu”) conformation, and the side chain of the so-called “gate” residue Tyr296 is consistently in the “gate-open” conformation. Among the wires tested, the most stable complex is produced when the wire has a −(CH_2)_4− linker. In this complex, the Ru(II)(phen)(bpy)_2 head group is level with the protein molecular surface. Crystal structures of AGAO in complex with optically pure forms of the C4 wire show that the linker and head group in the two enantiomers occupy slightly different positions in the active-site channel. Both the Λ and Δ isomers are effective competitive inhibitors of amine oxidation. Remarkably, inhibition by the C4 wire shows a high degree of selectivity for AGAO in comparison with other copper-containing amine oxidases.
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intramolecular electron transfer rate between active site copper and tpq in Arthrobacter Globiformis amine oxidase
Journal of Biological Inorganic Chemistry, 2006Co-Authors: Eric M. Shepard, David M. DooleyAbstract:Copper amine oxidases catalyze the oxidative deamination of primary amines operating through a ping-pong bi bi mechanism, divided into reductive and oxidative half-reactions. Considerable debate still exists regarding the role of copper in the oxidative half-reaction, where O2 is reduced to H2O2. Substrate-reduced amine oxidases display an equilibrium between a Cu(II) aminoquinol and a Cu(I) semiquinone, with the magnitude of the equilibrium constant being dependent upon the enzyme source. The initial electron transfer to dioxygen has been proposed to occur from either the reduced Cu(I) center or the reduced aminoquinol cofactor. In order for Cu(I) to be involved, it must be shown that the rate of electron transfer (kET) between the aminoquinol and Cu(II) is sufficiently rapid to place the Cu(I) semiquinone moiety on the mechanistic pathway. To further explore this issue, we measured the intramolecular electron transfer rate for the Cu(II) aminoquinol ⇆ Cu(I) semiquinone equilibrium in Arthrobacter Globiformis amine oxidase (AGAO) by temperature-jump relaxation techniques. The results presented herein establish that kET is greater than the rate of catalysis (kcat) for the preferred amine substrate β-phenylethylamine at three pH values, thereby permitting the Cu(I) semiquinone to be a viable catalytic intermediate during enzymatic reoxidation in this enzyme. The data show that kET is approximately equivalent at pH 6.2 and 7.2, being 2.5 times kcat for these pH values. At pH 8.2, however, kET decreases, becoming comparable to kcat. Potential reasons for the decreased kET at basic pH are presented. The implications of these results in light of a previously published study measuring reoxidation rates of substrate-reduced AGAO are also addressed.
Katsuyuki Tanizawa - One of the best experts on this subject based on the ideXlab platform.
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probing the catalytic mechanism of copper amine oxidase from Arthrobacter Globiformis with halide ions
Journal of Biological Chemistry, 2015Co-Authors: Takeshi Murakawa, Katsuyuki Tanizawa, Akio Hamaguchi, Shota Nakanishi, Misumi Kataoka, Tadashi Nakai, Yoshiaki Kawano, Hiroshi Yamaguchi, Hideyuki Hayashi, Toshihide OkajimaAbstract:The catalytic reaction of copper amine oxidase proceeds through a ping-pong mechanism comprising two half-reactions. In the initial half-reaction, the substrate amine reduces the Tyr-derived cofactor, topa quinone (TPQ), to an aminoresorcinol form (TPQamr) that is in equilibrium with a semiquinone radical (TPQsq) via an intramolecular electron transfer to the active-site copper. We have analyzed this reductive half-reaction in crystals of the copper amine oxidase from Arthrobacter Globiformis. Anerobic soaking of the crystals with an amine substrate shifted the equilibrium toward TPQsq in an "on-copper" conformation, in which the 4-OH group ligated axially to the copper center, which was probably reduced to Cu(I). When the crystals were soaked with substrate in the presence of halide ions, which act as uncompetitive and noncompetitive inhibitors with respect to the amine substrate and dioxygen, respectively, the equilibrium in the crystals shifted toward the "off-copper" conformation of TPQamr. The halide ion was bound to the axial position of the copper center, thereby preventing TPQamr from adopting the on-copper conformation. Furthermore, transient kinetic analyses in the presence of viscogen (glycerol) revealed that only the rate constant in the step of TPQamr/TPQsq interconversion is markedly affected by the viscogen, which probably perturbs the conformational change. These findings unequivocally demonstrate that TPQ undergoes large conformational changes during the reductive half-reaction.
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high resolution crystal structure of copper amine oxidase from Arthrobacter Globiformis assignment of bound diatomic molecules as o2
Acta Crystallographica Section D-biological Crystallography, 2013Co-Authors: Takeshi Murakawa, Katsuyuki Tanizawa, Hideyuki Hayashi, Tomoko Sunami, Kazuo Kurihara, Taro Tamada, Ryota Kuroki, Mamoru Suzuki, Toshihide OkajimaAbstract:The crystal structure of a copper amine oxidase from Arthrobacter Globiformis was determined at 1.08 A resolution with the use of low-molecular-weight polyethylene glycol (LMW PEG; average molecular weight ∼200) as a cryoprotectant. The final crystallographic R factor and Rfree were 13.0 and 15.0%, respectively. Several molecules of LMW PEG were found to occupy cavities in the protein interior, including the active site, which resulted in a marked reduction in the overall B factor and consequently led to a subatomic resolution structure for a relatively large protein with a monomer molecular weight of ∼70 000. About 40% of the presumed H atoms were observed as clear electron densities in the Fo − Fc difference map. Multiple minor conformers were also identified for many residues. Anisotropic displacement fluctuations were evaluated in the active site, which contains a post-translationally derived quinone cofactor and a Cu atom. Furthermore, diatomic molecules, most likely to be molecular oxygen, are bound to the protein, one of which is located in a region that had previously been proposed as an entry route for the dioxygen substrate from the central cavity of the dimer interface to the active site.
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crystal structures of the copper containing amine oxidase from Arthrobacter Globiformis in the holo and apo forms implications for the biogenesis of topaquinone
Biochemistry, 1998Co-Authors: M C J Wilce, David M. Dooley, Katsuyuki Tanizawa, H C Freeman, J M Guss, Hideyuki Matsunami, William S Mcintire, C E Ruggiero, H YamaguchiAbstract:The crystal structures of the copper enzyme phenylethylamine oxidase from the Gram-positive bacterium Arthrobacter Globiformis (AGAO) have been determined and refined for three forms of the enzyme: the holoenzyme in its active form (at 2.2 A resolution), the holoenzyme in an inactive form (at 2.8 A resolution), and the apoenzyme (at 2.2 A resolution). The holoenzyme has a topaquinone (TPQ) cofactor formed from the apoenzyme by the post-translational modification of a tyrosine residue in the presence of Cu2+. Significant differences between the three forms of AGAO are limited to the active site. The polypeptide fold is closely similar to those of the amine oxidases from Escherichia coli [Parsons, M. R., et al. (1995) Structure 3, 1171−1184] and pea seedlings [Kumar, V., et al. (1996) Structure 4, 943−955]. In the active form of holo-AGAO, the active-site Cu atom is coordinated by three His residues and two water molecules in an approximately square-pyramidal arrangement. In the inactive form, the Cu atom ...
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purification and properties of phenylethylamine oxidase of Arthrobacter Globiformis
Bioscience Biotechnology and Biochemistry, 1997Co-Authors: Eiichi Shimizu, Katsuyuki Tanizawa, Keiichi Ohta, Shigeo Takayama, Yuzuru Kitagaki, Takamitsu YorifujiAbstract:Phenylethylamine oxidase (EC class 1.4.3) of Arthrobacter Globiformis IFO 12137 (ATCC 8010) was purified to homogeneity. The enzyme had a Mr of 141,000 and was composed of two apparently identical subunits, which had a Mr of 71,000 and contained one copper ion. The absorption spectrum of the enzyme had maxima at 280 and 480nm, and the ratio A280/A480 was 61.5. Hydrogen peroxide was formed in the oxidation of amines. The enzyme was most active and stable at pH 6.5. 2-Phenylethylamine and tyramine were the most active substrates. Several aromatic monoamines, aliphatic monoamines with 4–11 carbons, higher aliphatic diamines, and histamine were poor substrates. Benzylamine, putrescine, spermine, and spermidine were not oxidized. The Kms for 2-phenylethylamine and tyramine were 18 and 85 μm, and the Vmaxs for them were 27.1 and 26.4μmol/min/mg of enzyme, respectively. Benzyl alcohol was a noncompetitive and benzylamine was a mix-type inhibitor of the enzyme. Carbonyl-blocking reagents such as methylhydrazine, ...
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copper topa quinone containing histamine oxidase from Arthrobacter Globiformis molecular cloning and sequencing overproduction of precursor enzyme and generation of topa quinone cofactor
Journal of Biological Chemistry, 1995Co-Authors: Yoonho Choi, Eiichi Shimizu, Takamitsu Yorifuji, R Matsuzaki, T Fukui, Hidetoshi Sato, Yukihiro Ozaki, Katsuyuki TanizawaAbstract:The gene coding for histamine oxidase has been cloned and sequenced from a Coryneform bacterium Arthrobacter Globiformis. The deduced amino acid sequence consists of 684 residues with a calculated molecular mass of 75,109 daltons and shows a high overall identity (58%) with that of phenethylamine oxidase derived from the same bacterial strain. Although the sequence similarities are rather low when compared with copper amine oxidases from other organisms, the consensus Asn-Tyr-Asp/Glu sequence, in which the middle Tyr is the precursor to the quinone cofactor (the quinone of 2,4,5-trihydroxyphenylalanine, topa) covalently bound to this class of enzymes, is also conserved in the histamine oxidase sequence. To identify the quinone cofactor, an overexpression plasmid has been constructed for the recombinant histamine oxidase. The inactive enzyme purified from the transformed Escherichia coli cells grown in a copper-depleted medium gained maximal activity upon stoichiometric binding of cupric ions. Concomitantly with the enzyme activation by copper, a brownish pink compound was generated in the enzyme, which was identified as the quinone of topa by absorption and resonance Raman spectroscopies of the p-nitrophenylhydrazine-derivatized enzyme and found at the position corresponding to the precursor Tyr (Tyr-402). Therefore, the copper-dependent autoxidation of a specific tyrosyl residue operates on the formation of the topa quinone cofactor in this enzyme, as recently demonstrated with the precursor form of phenethylamine oxidase (Matsuzaki, R., Fukui, T., Sato, H., Ozaki, Y., and Tanizawa, K.(1994) FEBS Lett. 351, 360-364).
Lawrence Que - One of the best experts on this subject based on the ideXlab platform.
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the role of histidine 200 in mndd the mn ii dependent 3 4 dihydroxyphenylacetate 2 3 dioxygenase from Arthrobacter Globiformis cm 2 a site directed mutagenesis study
Journal of Biological Inorganic Chemistry, 2005Co-Authors: Joseph P Emerson, Michael J. Sadowsky, Lawrence Que, M L Wagner, Mark F Reynolds, Lawrence P. WackettAbstract:The manganese-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase (MndD) from Arthrobacter Globiformis CM-2 is an extradiol-cleaving catechol dioxygenase that catalyzes aromatic ring cleavage of 3,4-dihydroxyphenylacetate (DHPA). Based on the recent crystal structure of the MndD–DHPA complex, a series of site-directed mutations were made at a conserved second-sphere residue, histidine 200, to gain insight into and clarify the role this residue plays in the Mn(II)-dependent catalytic mechanism. In this study, we report the activities and spectroscopic data of these H200 variants and their DHPA and 4-nitrocatechol (4-NC) complexes. The data collected from wild-type and mutant MndDs are consistent with a role for H200 interacting with a manganese-bound dioxygen moiety and are inconsistent with other previously proposed roles involving proton transfer. Spectroscopic observations, including unique low-field EPR signals found when DHPA and 4-NC are bound to the Mn(II) center of MndD, are discussed and their relationship to dioxygen activation catalyzed in MndD is explored.
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manganese ii active site mutants of 3 4 dihydroxyphenylacetate 2 3 dioxygenase from Arthrobacter Globiformis strain cm 2
Biochemistry, 1997Co-Authors: Yvonne R. Boldt, Michael J. Sadowsky, Lawrence Que, Adam K Whiting, M L Wagner, Lawrence P. WackettAbstract:Whereas all other members of the extradiol-cleaving catechol dioxygenase family are iron-dependent, the 3,4-dihydroxyphenylacetate 2,3-dioxygenase (MndD) from Arthrobacter Globiformis CM-2 is dependent on manganese for catalytic activity. Recently, the endogenous iron ligands of one family member, the 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC), were identified crystallographically as two histidines and a glutamic acid [Sugiyama, K., et al. (1995) Proc. Jpn. Acad., Ser. B 71, 32-35; Han, et al. (1995) Science 270, 976-980; Senda, T., et al. (1996) J. Mol. Biol. 255, 735-752]. Though BphC and MndD have low overall sequence identity (23%), the three BphC metal ligands are all conserved in MndD (H155, H214, and E266). In order to determine whether these residues also act as ligands to manganese in MndD, site-directed mutants of each were constructed, purified, and analyzed for activity and metal content. Mutations H155A, H214A, and E266Q yielded purified enzymes with specific activities of <0.1% of that of the wild-type dioxygenase and bound 0.4, 1.8, and 33% of the wild-type level of manganese, respectively. The relatively high level of manganese [with a Mn(II) EPR signal distinctly different from that of the wild-type enzyme] observed for E266Q suggests that the glutamine may act as a weak ligand to the metal. Mutant E266D, which retains the potential metal binding capability of a carboxylate group, exhibited 12% of the wild-type activity in crude extracts, suggesting that Mn remains bound; however, this mutant protein was too unstable to be purified and analyzed for metal content. On the basis of the low activity and metal content of mutant proteins, we propose that the conserved residues H155, H214, and E266 ligate manganese in MndD. As is the case with the superoxide dismutases, the extradiol-cleaving catechol dioxygenases appear to utilize identical coordinating residues for their iron- and manganese-dependent enzymes.
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manganese ii dependent extradiol cleaving catechol dioxygenase from Arthrobacter Globiformis cm 2
Biochemistry, 1996Co-Authors: Adam K Whiting, Yvonne R. Boldt, Lawrence P. Wackett, Michael P Hendrich, Lawrence QueAbstract:A manganese-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase from Arthrobacter Globiformis strain CM-2 (MndD) cloned in Escherichia coli has been purified to homogeneity. Sedimentation equilibrium analysis indicates an α4 homotetrameric holoenzyme structure (4 × 38 861 Da). Steady-state kinetic analysis of MndD with a variety of substrates and inhibitors yields very similar relative rates to the known Fe(II)- and Mn(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenases from Pseudomonas ovalis and Bacillus brevis, respectively. Yet, unlike the Fe(II)-dependent enzyme, MndD retains almost all activity in the presence of H2O2 and CN- and is inactivated by Fe(II). ICP emission analysis confirms the presence of 3.0 ± 0.2 g-atoms Mn (and only 0.7 ± 0.2 g-atoms Fe) per tetrameric holoenzyme molecule. Comparison of MndD samples with varying metal content, including an apo and partial-apo enzyme preparation, shows a strong positive correlation between specific activity and Mn content. EPR spectra of MndD a...
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A manganese-dependent dioxygenase from Arthrobacter Globiformis CM-2 belongs to the major extradiol dioxygenase family.
Journal of bacteriology, 1995Co-Authors: Yvonne R. Boldt, Michael J. Sadowsky, L. B. M. Ellis, Lawrence Que, Lawrence P. WackettAbstract:Almost all bacterial ring cleavage dioxygenases contain iron as the catalytic metal center. We report here the first available sequence for a manganese-dependent 3,4-dihydroxyphenylacetate (3,4-DHPA) 2,3-dioxygenase and its further characterization. This manganese-dependent extradiol dioxygenase from Arthrobacter Globiformis CM-2, unlike iron-dependent extradiol dioxygenases, is not inactivated by hydrogen peroxide. Also, ferrous ions, which activate iron extradiol dioxygenases, inhibit 3,4-DHPA 2,3-dioxygenase. The gene encoding 3,4-DHPA 2,3-dioxygenase, mndD, was identified from an A. Globiformis CM-2 cosmid library. mndD was subcloned as a 2.0-kb SmaI fragment in pUC18, from which manganese-dependent extradiol dioxygenase activity was expressed at high levels in Escherichia coli. The mndD open reading frame was identified by comparison with the known N-terminal amino acid sequence of purified manganese-dependent 3,4-DHPA 2,3-dioxygenase. Fourteen of 18 amino acids conserved in members of the iron-dependent extradiol dioxygenase family are also conserved in the manganese-dependent 3,4-DHPA 2,3-dioxygenase (MndD). Thus, MndD belongs to the extradiol family of dioxygenases and may share a common ancestry with the iron-dependent extradiol dioxygenases. We propose the revised consensus primary sequence (G,T,N,R)X(H,A)XXXXXXX(L,I,V,M,F)YXX(D,E,T,N,A)PX(G,P) X(2,3)E for this family. (Numbers in brackets indicate a gap of two or three residues at this point in the sequence.) The suggested common ancestry is also supported by sequence obtained from genes flanking mndD, which share significant sequence identity with xylJ and xylG from Pseudomonas putida.
Yvonne R. Boldt - One of the best experts on this subject based on the ideXlab platform.
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manganese ii active site mutants of 3 4 dihydroxyphenylacetate 2 3 dioxygenase from Arthrobacter Globiformis strain cm 2
Biochemistry, 1997Co-Authors: Yvonne R. Boldt, Michael J. Sadowsky, Lawrence Que, Adam K Whiting, M L Wagner, Lawrence P. WackettAbstract:Whereas all other members of the extradiol-cleaving catechol dioxygenase family are iron-dependent, the 3,4-dihydroxyphenylacetate 2,3-dioxygenase (MndD) from Arthrobacter Globiformis CM-2 is dependent on manganese for catalytic activity. Recently, the endogenous iron ligands of one family member, the 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC), were identified crystallographically as two histidines and a glutamic acid [Sugiyama, K., et al. (1995) Proc. Jpn. Acad., Ser. B 71, 32-35; Han, et al. (1995) Science 270, 976-980; Senda, T., et al. (1996) J. Mol. Biol. 255, 735-752]. Though BphC and MndD have low overall sequence identity (23%), the three BphC metal ligands are all conserved in MndD (H155, H214, and E266). In order to determine whether these residues also act as ligands to manganese in MndD, site-directed mutants of each were constructed, purified, and analyzed for activity and metal content. Mutations H155A, H214A, and E266Q yielded purified enzymes with specific activities of <0.1% of that of the wild-type dioxygenase and bound 0.4, 1.8, and 33% of the wild-type level of manganese, respectively. The relatively high level of manganese [with a Mn(II) EPR signal distinctly different from that of the wild-type enzyme] observed for E266Q suggests that the glutamine may act as a weak ligand to the metal. Mutant E266D, which retains the potential metal binding capability of a carboxylate group, exhibited 12% of the wild-type activity in crude extracts, suggesting that Mn remains bound; however, this mutant protein was too unstable to be purified and analyzed for metal content. On the basis of the low activity and metal content of mutant proteins, we propose that the conserved residues H155, H214, and E266 ligate manganese in MndD. As is the case with the superoxide dismutases, the extradiol-cleaving catechol dioxygenases appear to utilize identical coordinating residues for their iron- and manganese-dependent enzymes.
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manganese ii dependent extradiol cleaving catechol dioxygenase from Arthrobacter Globiformis cm 2
Biochemistry, 1996Co-Authors: Adam K Whiting, Yvonne R. Boldt, Lawrence P. Wackett, Michael P Hendrich, Lawrence QueAbstract:A manganese-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase from Arthrobacter Globiformis strain CM-2 (MndD) cloned in Escherichia coli has been purified to homogeneity. Sedimentation equilibrium analysis indicates an α4 homotetrameric holoenzyme structure (4 × 38 861 Da). Steady-state kinetic analysis of MndD with a variety of substrates and inhibitors yields very similar relative rates to the known Fe(II)- and Mn(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenases from Pseudomonas ovalis and Bacillus brevis, respectively. Yet, unlike the Fe(II)-dependent enzyme, MndD retains almost all activity in the presence of H2O2 and CN- and is inactivated by Fe(II). ICP emission analysis confirms the presence of 3.0 ± 0.2 g-atoms Mn (and only 0.7 ± 0.2 g-atoms Fe) per tetrameric holoenzyme molecule. Comparison of MndD samples with varying metal content, including an apo and partial-apo enzyme preparation, shows a strong positive correlation between specific activity and Mn content. EPR spectra of MndD a...
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A manganese-dependent dioxygenase from Arthrobacter Globiformis CM-2 belongs to the major extradiol dioxygenase family.
Journal of bacteriology, 1995Co-Authors: Yvonne R. Boldt, Michael J. Sadowsky, L. B. M. Ellis, Lawrence Que, Lawrence P. WackettAbstract:Almost all bacterial ring cleavage dioxygenases contain iron as the catalytic metal center. We report here the first available sequence for a manganese-dependent 3,4-dihydroxyphenylacetate (3,4-DHPA) 2,3-dioxygenase and its further characterization. This manganese-dependent extradiol dioxygenase from Arthrobacter Globiformis CM-2, unlike iron-dependent extradiol dioxygenases, is not inactivated by hydrogen peroxide. Also, ferrous ions, which activate iron extradiol dioxygenases, inhibit 3,4-DHPA 2,3-dioxygenase. The gene encoding 3,4-DHPA 2,3-dioxygenase, mndD, was identified from an A. Globiformis CM-2 cosmid library. mndD was subcloned as a 2.0-kb SmaI fragment in pUC18, from which manganese-dependent extradiol dioxygenase activity was expressed at high levels in Escherichia coli. The mndD open reading frame was identified by comparison with the known N-terminal amino acid sequence of purified manganese-dependent 3,4-DHPA 2,3-dioxygenase. Fourteen of 18 amino acids conserved in members of the iron-dependent extradiol dioxygenase family are also conserved in the manganese-dependent 3,4-DHPA 2,3-dioxygenase (MndD). Thus, MndD belongs to the extradiol family of dioxygenases and may share a common ancestry with the iron-dependent extradiol dioxygenases. We propose the revised consensus primary sequence (G,T,N,R)X(H,A)XXXXXXX(L,I,V,M,F)YXX(D,E,T,N,A)PX(G,P) X(2,3)E for this family. (Numbers in brackets indicate a gap of two or three residues at this point in the sequence.) The suggested common ancestry is also supported by sequence obtained from genes flanking mndD, which share significant sequence identity with xylJ and xylG from Pseudomonas putida.
