X-ray Absorption Studies on Catechol 2,3-Dioxygenase from Pseudomonas putida MT2

Bertini, Ivano; Briganti, Fabrizio; Mangani, Stefano; Nolting, H.‐F.; Scozzafava, A.

doi:10.1021/bi00201a027

Cited by 36 publications

(22 citation statements)

References 41 publications

(51 reference statements)

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“…The four residues that we have identified as ligating the active site metal in both FeARD' and NiARD isoforms, His 96, His 98, Asp 102 and His 140, are all strictly conserved across the ARD superfamily (31), and were initially identified as potential ligands via sequence alignment and spatial localization from paramagnetic broadening patterns in NMR data and homology modeling (12). The recently-published crystal structure of MmARD supports the assignment of these residues as the protein-based ligands (17).…”

Section: Discussionmentioning

confidence: 93%

Characterization of Metal Binding in the Active Sites of Acireductone Dioxygenase Isoforms from Klebsiella ATCC 8724

Chai

Dang

et al. 2008

Biochemistry

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The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella ATCC 8724 present an unusual case in which two enzymes with different structures and distinct activities towards their common substrates (1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene and dioxygen) are derived from the same polypeptide chain. Structural and functional differences between the two isozymes are determined by the type of M +2 metal ion bound in the active site. The Ni +2 -bound NiARD catalyzes an off-pathway shunt from the methionine salvage pathway leading to the production of formate, methylthiopropionate and carbon monoxide, while the Fe +2 -bound FeARD' catalyzes the on-pathway formation of methionine precursor 2-keto-4-methylthiobutyrate and formate. Four potential protein-based metal ligands were identified by sequence homology and structural considerations. Based on the results of site-directed mutagenesis experiments, X-ray absorption spectroscopy (XAS) and isothermal calorimetry measurements, it is concluded that the same four residues, His 96, His 98, Glu 102 and His 140, provide the protein-based ligands for the metal in both the Ni-and Fe-containing forms of the enzyme, and subtle differences in the local backbone conformations trigger the observed structural and functional differences between the FeARD' and NiARD isozymes. Furthermore, both forms of the enzyme bind their respective metals with pseudo-octahedral geometry, and both may lose a His ligand upon binding of substrate under anaerobic conditions. However, mutations at two conserved non-ligand acidic residues, Glu 95 and Glu 100, result in low metal contents for the mutant proteins as isolated, suggesting that some of the conserved charged residues may aid in transfer of metal from in vivo sources or prevent the loss of metal to stronger chelators. The Glu 100 mutant reconstitutes readily but has low activity. Mutation of Asp 101 results in an active enzyme that incorporates metal in vivo but shows evidence of mixed forms.* To whom correspondence should be addressed: pochapsk@brandeis.edu, phone 781−736−2559, fax 781−736−2516. e Current address: Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, 70 Ship Street, GE3, Providence, RI, 02912 f Current address: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Dr., Room 1007, Indianapolis IN 46202 g This work was supported in part by USPHS grant (R01-GM067786 TCP). Acknowledgment is made to the donors of The American Chemical Society Petroleum Research Fund for partial support (MJM). Trainee funding (SCC) was provided by the NIH-Chemistry Biology Interface Program, T32-GM08515. XAS data collection at the National Synchrotron Light Source at Brookhaven National Laboratory was supported by the U.S. Department of Energy, Division of Materials Sciences and Division of Chemical Sciences. Beamline X9B at NSLS is supported in part by the NIH.Supporting information available Fits for resting state and ES...

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Section: Discussionmentioning

confidence: 93%

Characterization of Metal Binding in the Active Sites of Acireductone Dioxygenase Isoforms from Klebsiella ATCC 8724

Chai

Dang

et al. 2008

Biochemistry

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“…The higher 1s → 3d peak area at pH 8.5 could be due to higher distortion of the octahedral site as a result of one shorter Fe-N/O bond. Typical 1s → 3d peak areas for octahedral geometries are 3 – 7 × 10 −2 eV,(49) whereas lowering symmetry to a five-coordinate geometry is associated with peak areas of 8 – 13 × 10 −2 eV (2, 3). The measured peak area for the high pH sample is at the high end of what is typically observed for six-coordinate Fe(II), but could reflect a six-coordinate site with deviations from ideal octahedral symmetry.…”

Section: Resultsmentioning

confidence: 99%

Inverse Solvent Isotope Effects Demonstrate Slow Aquo Release from Hypoxia Inducible Factor-Prolyl Hydroxylase (PHD2)

et al. 2012

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Prolyl Hydroxylase Domain 2 (PHD2) is deemed a primary oxygen sensor in humans yet many details of its underlying mechanism are still not fully understood. (Fe2++αKG)PHD2 is 6-coordinate, with a 2His/1Asp facial triad occupying 3 coordination sites, a bidentate α-ketoglutarate occupying two sites and an aquo ligand in the final site. Turnover is thought to be initiated upon release of the aquo ligand, creating a site for O2 to bind at the iron. Herein we show that steady-state turnover is faster under acidic conditions, with kcat exhibiting a kinetic pKa = 7.22. A variety of spectroscopic probes were employed to identify the active-site acid, through comparison of (Fe2++αKG)PHD2 at pH 6.50 with pH 8.50. The near-UV circular dichroism spectrum was virtually unchanged at elevated pH, indicating that the secondary structure did not change as a function of pH. UV-visible and Fe X-ray absorption spectroscopy indicated that the primary coordination sphere of Fe2+ changed upon increasing the pH; EXAFS analysis found a short Fe-(O/N) bond length of 1.96 Å at pH 8.50, strongly suggesting that the aquo ligand was deprotonated at this pH. Solvent isotope effects were measured during steady-sate turnover over a wide pH-range, with an inverse SIE of on kcat observed (D2Okcat = 0.91 ± 0.03) for the acid form; a similar SIE was observed for the basic form of enzyme (D2Okcat = 0.9 ± 0.1), with an acid equilibrium offset of ΔpKa = 0.67 ± 0.04. The inverse SIE indicated that aquo release from the active site Fe2+ immediately precedes a rate-limiting step, suggesting that turnover in this enzyme may be partially limited by the rate of O2 binding or activation, and suggesting that aquo release is relatively slow. The unusual kinetic pKa further suggested that PHD2 might function physiologically to sense both intracellular pO2 as well as pH, which could provide for feedback between anaerobic metabolism and hypoxia sensing.

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“…This region contains one histidine residue, two aspartate residues, and one glutamate residue. For the catechol 2,3-DO from P. putida mt-2, it has been recently suggested that histidine and carboxylate groups from aspartate and/or glutamate residues are responsible for iron(II) coordination (8). It therefore seems reasonable that this region is in the three proteins involved in the coordination of the ferrous iron.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a 2,3-dihydroxybiphenyl dioxygenase from the naphthalenesulfonate-degrading bacterium strain BN6

et al. 1995

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An extradiol dioxygenase was cloned from the naphthalenesulfonate-degrading bacterial strain BN6 by screening a gene bank for colonies with 2,3-dihydroxybiphenyl dioxygenase activity. DNA sequence analysis of a 1,358-bp fragment revealed an open reading frame of only 486 bp. This is the smallest gene encoding an extradiol dioxygenase found until now. Expression of the gene in a T7 expression vector enabled purification of the enzyme. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the protein was a dimer with a subunit size of 21.7 kDa. The enzyme oxidized 2,3-dihydroxybiphenyl, 3-isopropylcatechol, 3-and 4-chlorocatechol, and 3-and 4-methylcatechol. Since the ability to convert 3-chlorocatechol is an unusual characteristic for an extradiol-cleaving dioxygenase, this reaction was analyzed in more detail. The deduced amino-terminal amino acid sequence differed from the corresponding sequence of the 1,2-dihydroxynaphthalene dioxygenase, which had been determined earlier from the enzyme purified from this strain. This indicates that strain BN6 carries at least two different extradiol dioxygenases.A bacterial strain (strain BN6) which is able to degrade amino-and hydroxynaphthalenesulfonates is currently being studied in this laboratory. This strain oxidizes substituted naphthalenesulfonates to the corresponding substituted 1,2-dihydroxynaphthalenes by a reaction catalyzed by a desulfonating dioxygenase. Subsequent metabolism of the substituted 1,2-dihydroxynaphthalenes (1,2-DHNs) to substituted salicylates follows the common naphthalene degradative pathway. Strain BN6 does not mineralize naphthalenesulfonates, since the salicylates are not further oxidized (24,25,33,34). Cloning the genes for the metabolism of these naphthalenesulfonates would facilitate mobilization into salicylate-degrading bacterial strains, thereby accomplishing the complete degradation of naphthalenesulfonates in a single strain. Previously, the 1,2-DHN dioxygenase (DHNDO), which is part of this metabolic pathway, was purified to homogeneity and biochemically characterized. The enzyme oxidized 1,2-DHN, 2,3-dihydroxybiphenyl (2,3-DHBP), and some other aromatic diols (25). In the present study, we attempted to clone the corresponding gene from strain BN6. Although we obtained clones derived from strain BN6 which were able to oxidize 2,3-DHBP, the encoded gene was unexpectedly not involved in the degradation of naphthalenesulfonates. The extradiol dioxygenase (DO) encoded by this clone was examined in the present study. MATERIALS AND METHODSBacterial strains and culture conditions. The isolation and characterization of strain BN6 have been described previously (33). The strain has been deposited at the Deutsche Sammlung von Mikroorganismen, Brunswick, Germany, as DSM 6383. For the isolation of genomic DNA, the strain was grown in nutrient broth. Escherichia coli DH5␣ was the host for construction of the genomic library. E. coli JM 109 was used for subcloning and isolation of DNA for sequencing. Fo...

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X-ray Absorption Studies on Catechol 2,3-Dioxygenase from Pseudomonas putida MT2

Cited by 36 publications

References 41 publications

Characterization of Metal Binding in the Active Sites of Acireductone Dioxygenase Isoforms from Klebsiella ATCC 8724

Characterization of Metal Binding in the Active Sites of Acireductone Dioxygenase Isoforms from Klebsiella ATCC 8724

Inverse Solvent Isotope Effects Demonstrate Slow Aquo Release from Hypoxia Inducible Factor-Prolyl Hydroxylase (PHD2)

Characterization of a 2,3-dihydroxybiphenyl dioxygenase from the naphthalenesulfonate-degrading bacterium strain BN6

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