The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
K. V. Rajagopalan - One of the best experts on this subject based on the ideXlab platform.
-
structure based alteration of substrate specificity and catalytic activity of Sulfite Oxidase from Sulfite oxidation to nitrate reduction
Biochemistry, 2012Co-Authors: James A Qiu, Heather L Wilson, K. V. RajagopalanAbstract:Eukaryotic Sulfite Oxidase is a dimeric protein that contains the molybdenum cofactor and catalyzes the metabolically essential conversion of Sulfite to sulfate as the terminal step in the metabolism of cysteine and methionine. Nitrate reductase is an evolutionarily related molybdoprotein in lower organisms that is essential for growth on nitrate. In this study, we describe human and chicken Sulfite Oxidase variants in which the active site has been modified to alter substrate specificity and activity from Sulfite oxidation to nitrate reduction. On the basis of sequence alignments and the known crystal structure of chicken Sulfite Oxidase, two residues are conserved in nitrate reductases that align with residues in the active site of Sulfite Oxidase. On the basis of the crystal structure of yeast nitrate reductase, both positions were mutated in human Sulfite Oxidase and chicken Sulfite Oxidase. The resulting double-mutant variants demonstrated a marked decrease in Sulfite Oxidase activity but gained nitrate reductase activity. An additional methionine residue in the active site was proposed to be important in nitrate catalysis, and therefore, the triple variant was also produced. The nitrate reducing ability of the human Sulfite Oxidase triple mutant was nearly 3-fold greater than that of the double mutant. To obtain detailed structural data for the active site of these variants, we introduced the analogous mutations into chicken Sulfite Oxidase to perform crystallographic analysis. The crystal structures of the Mo domains of the double and triple mutants were determined to 2.4 and 2.1 A resolution, respectively.
-
The structures of the C185S and C185A mutants of Sulfite Oxidase reveal rearrangement of the active site.
Biochemistry, 2010Co-Authors: James A Qiu, Graham N George, Heather L Wilson, Caroline Kisker, M. Jake Pushie, K. V. RajagopalanAbstract:Sulfite Oxidase (SO) catalyzes the physiologically critical conversion of Sulfite to sulfate. Enzymatic activity is dependent on the presence of the metal molybdenum complexed with a pyranopterin-dithiolene cofactor termed molybdopterin. Comparison of the amino acid sequences of SOs from a variety of sources has identified a single conserved Cys residue essential for catalytic activity. The crystal structure of chicken liver Sulfite Oxidase indicated that this residue, Cys185 in chicken SO, coordinates the Mo atom in the active site. To improve our understanding of the role of this residue in the catalytic mechanism of Sulfite Oxidase, serine and alanine variants at position 185 of recombinant chicken SO were generated. Spectroscopic and kinetic studies indicate that neither variant is capable of Sulfite oxidation. The crystal structure of the C185S variant was determined to 1.9 A resolution and to 2.4 A resolution in the presence of Sulfite, and the C185A variant to 2.8 A resolution. The structures of th...
-
electrocatalytically functional multilayer assembly of Sulfite Oxidase and cytochrome c
Soft Matter, 2008Co-Authors: Roberto Spricigo, Frieder W. Scheller, Silke Leimkühler, K. V. Rajagopalan, Roman Dronov, Fred Lisdat, Ulla WollenbergerAbstract:An electrocatalytically functional multilayer has been designed using two proteins, cytochrome c and Sulfite Oxidase, and a polyelectrolyte (polyaniline sulfonate). The two proteins were co-immobilized on the surface of a gold electrode in alternating layers by electrostatic interactions using the layer-by-layer technique. The formation of this fully electro-active multilayer is characterized by quartz crystal microbalance and electrochemical experiments. The electro-catalytic characterization of the device containing up to 12 layers is based on generation of an oxidation current after Sulfite addition. Besides the electron-transfer mechanism, the role of the different components in the electron-transport chain is clarified. Kinetic data were extracted to characterize the multilayer function. This artificial multilayer assembly is expected to be useful in the biosensor and biofuel cell development.
-
layer by layer arrangement by protein protein interaction of Sulfite Oxidase and cytochrome c catalyzing oxidation of Sulfite
Journal of the American Chemical Society, 2008Co-Authors: Roman Dronov, Frieder W. Scheller, Roberto Spricigo, Ulla Wollenberger, K. V. Rajagopalan, Dirk G Kurth, Helmuth Mohwald, S Leimkuehler, Fred LisdatAbstract:Layer-by-layer self-assembly of Sulfite Oxidase and cytochrome c was carried out without additional polymeric polyelectrolytes. The arrangement shows a linear increase in the immobilized protein mass after each deposition cycle. The modified electrodes demonstrate electrocatalytic activity for Sulfite oxidation generating catalytic current with a linear increase with the number of layers. This effect shows that, in the protein assembly without a polyelectrolyte, electron transfer occurs, thus supporting the concept for direct interprotein electron exchange.
-
modified active site coordination in a clinical mutant of Sulfite Oxidase
Journal of the American Chemical Society, 2007Co-Authors: Christian J Doonan, Robert M. Garrett, K. V. Rajagopalan, Heather L Wilson, Brian Bennett, Roger C Prince, Graham N GeorgeAbstract:The molybdenum site of the Arginine 160 {yields} Glutamine clinical mutant of the physiologically vital enzyme Sulfite Oxidase has been investigated by a combination of X-ray absorption spectroscopy and density functional theory calculations. We conclude that the mutant enzyme has a six-coordinate pseudo-octahedral active site with coordination of Glutamine O{sup {epsilon}} to molybdenum. This contrasts with the wild-type enzyme which is five-coordinate with approximately square-based pyramidal geometry. This difference in the structure of the molybdenum site explains many of the properties of the mutant enzyme which have previously been reported.
John H. Enemark - One of the best experts on this subject based on the ideXlab platform.
-
kinetic results for mutations of conserved residues h304 and r309 of human Sulfite Oxidase point to mechanistic complexities
Metallomics, 2014Co-Authors: Amanda C. Davis, Gordon Tollin, Kayunta Johnsonwinters, Anna R Arnold, John H. EnemarkAbstract:Several point mutations in the gene of human Sulfite Oxidase (hSO) result in isolated Sulfite Oxidase deficiency, an inherited metabolic disorder. Three conserved residues (H304, R309, K322) are hydrogen bonded to the phosphate group of the molybdenum cofactor, and the R309H and K322R mutations are responsible for isolated Sulfite Oxidase deficiency. The kinetic effects of the K322R mutation have been previously reported (Rajapakshe et al., Chem. Biodiversity, 2012, 9, 1621–1634); here we investigate several mutants of H304 and R309 by steady-state kinetics, laser flash photolysis studies of intramolecular electron transfer (IET), and spectroelectrochemistry. An unexpected result is that all of the mutants show decreased rates of IET but increased steady-state rates of catalysis. However, in all cases the rate of IET is greater than the overall turnover rate, showing that IET is not the rate determining step for any of the mutations.
-
Pulsed Electron Paramagnetic Resonance Spectroscopy of 33S-Labeled Molybdenum Cofactor in Catalytically Active Bioengineered Sulfite Oxidase
Inorganic chemistry, 2014Co-Authors: Eric L. Klein, Gunter Schwarz, Abdel A. Belaidi, Arnold M. Raitsimring, Amanda C. Davis, Tobias Krämer, Andrei V. Astashkin, Frank Neese, John H. EnemarkAbstract:Molybdenum enzymes contain at least one pyranopterin dithiolate (molybdopterin, MPT) moiety that coordinates Mo through two dithiolate (dithiolene) sulfur atoms. For Sulfite Oxidase (SO), hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) of magnetic nuclei (I ≠ 0) near the Mo(V) (d1) center have been measured using high-resolution pulsed electron paramagnetic resonance (EPR) methods and interpreted with the help of density functional theory (DFT) calculations. These have provided important insights about the active site structure and the reaction mechanism of the enzyme. However, it has not been possible to use EPR to probe the dithiolene sulfurs directly since naturally abundant 32S has no nuclear spin (I = 0). Here we describe direct incorporation of 33S (I = 3/2), the only stable magnetic sulfur isotope, into MPT using controlled in vitro synthesis with purified proteins. The electron spin echo envelope modulation (ESEEM) spectra from 33S-labeled MPT in this catalytically active SO...
-
identity of the exchangeable sulfur containing ligand at the mo v center of r160q human Sulfite Oxidase
Inorganic Chemistry, 2012Co-Authors: Eric L. Klein, Arnold M. Raitsimring, Andrei V. Astashkin, Kayunta Johnsonwinters, Anna R Arnold, Asha Rajapakshe, Alexei Potapov, Daniella Goldfarb, John H. EnemarkAbstract:In our previous study of the fatal R160Q mutant of human Sulfite Oxidase (hSO) at low pH (Astashkin et al. J. Am. Chem. Soc.2008, 130, 8471–8480), a new Mo(V) species, denoted “species 1”, was observed at low pH values. Species 1 was ascribed to a six-coordinate Mo(V) center with an exchangeable terminal oxo ligand and an equatorial sulfate group on the basis of pulsed EPR spectroscopy and 33S and 17O labeling. Here we report new results for species 1 of R160Q, based on substitution of the sulfur-containing ligand by a phosphate group, pulsed EPR spectroscopy in Ka- and W-bands, and extensive density functional theory (DFT) calculations applied to large, more realistic molecular models of the enzyme active site. The combined results unambiguously show that species 1 has an equatorial Sulfite as the only exchangeable ligand. The two types of 17O signals that are observed arise from the coordinated and remote oxygen atoms of the Sulfite ligand. A typical five-coordinate Mo(V) site is compatible with the obs...
-
effects of large scale amino acid substitution in the polypeptide tether connecting the heme and molybdenum domains on catalysis in human Sulfite Oxidase
Metallomics, 2010Co-Authors: Kayunta Johnsonwinters, Gordon Tollin, Amanda C. Davis, Anna R Nordstrom, John H. EnemarkAbstract:Sulfite Oxidase (SO) is a molybdenum-cofactor-dependent enzyme that catalyzes the oxidation of Sulfite to sulfate, the final step in the catabolism of the sulfur-containing amino acids, cysteine and methionine. The catalytic mechanism of vertebrate SO involves intramolecular electron transfer (IET) from molybdenum to the integral b-type heme of SO and then to exogenous cytochrome c. However, the crystal structure of chicken Sulfite Oxidase (CSO) has shown that there is a 32 A distance between the Fe and Mo atoms of the respective heme and molybdenum domains, which are connected by a flexible polypeptide tether. This distance is too long to be consistent with the measured IET rates. Previous studies have shown that IET is viscosity dependent (Feng et al., Biochemistry, 2002, 41, 5816) and also dependent upon the flexibility and length of the tether (Johnson-Winters et al., Biochemistry, 2010, 49, 1290). Since IET in CSO is more rapid than in human Sulfite Oxidase (HSO) (Feng et al., Biochemistry, 2003, 42, 12235) the tether sequence of HSO has been mutated into that of CSO, and the resultant chimeric HSO enzyme investigated by laser flash photolysis and steady-state kinetics in order to study the specificity of the tether sequence of SO on the kinetic properties. Surprisingly, the IET kinetics of the chimeric HSO protein with the CSO tether sequence are slower than wildtype HSO. This observation raises the possibility that the composition of the non-conserved tether sequence of animal SOs may be optimized for individual species.
-
effects of interdomain tether length and flexibility on the kinetics of intramolecular electron transfer in human Sulfite Oxidase
Biochemistry, 2010Co-Authors: Kayunta Johnsonwinters, Andrei V. Astashkin, Gordon Tollin, Anna R Nordstrom, Safia Emesh, Asha Rajapakshe, Robert E Berry, John H. EnemarkAbstract:Sulfite Oxidase (SO) is a vitally important molybdenum enzyme that catalyzes the oxidation of toxic Sulfite to sulfate. The proposed catalytic mechanism of vertebrate SO involves two intramolecular one-electron transfer (IET) steps from the molybdenum cofactor to the iron of the integral b-type heme and two intermolecular one-electron steps to exogenous cytochrome c. In the crystal structure of chicken SO [Kisker, C., et al. (1997) Cell 91, 973−983], which is highly homologous to human SO (HSO), the heme iron and molybdenum centers are separated by 32 A and the domains containing these centers are linked by a flexible polypeptide tether. Conformational changes that bring these two centers into greater proximity have been proposed [Feng, C., et al. (2003) Biochemistry 42, 5816−5821] to explain the relatively rapid IET kinetics, which are much faster than those theoretically predicted from the crystal structure. To explore the proposed role(s) of the tether in facilitating this conformational change, we alt...
Ulla Wollenberger - One of the best experts on this subject based on the ideXlab platform.
-
three dimensional Sulfite Oxidase bioanodes basedon graphene functionalized carbon paper for Sulfite o2 biofuelcells
ACS Catalysis, 2019Co-Authors: Jing Tang, Silke Leimkühler, Ulla Wollenberger, Rebecka Maria Larsen Werchmeister, Loredana Preda, Wei Huang, Zhiyong Zheng, Xinxin Xiao, Christian Engelbrekt, Jens UlstrupAbstract:We have developed a three-dimensional (3D) graphene electrode suitable for the immobilization of human Sulfite Oxidase (hSO), which catalyzes the electrochemical oxidation of Sulfite via direct electron transfer (DET). The electrode is fabricated by drop-casting graphene-polyethylenimine (G-P) composites on carbon papers (CPs) precoated with graphene oxide (GO). The negatively charged hSO can be adsorbed electrostatically on the positively charged matrix (G-P) on CP electrodes coated with GO (CPG), with a proper orientation for accelerated DET. Notably, further electrochemical reduction of G-P on CPG electrodes leads to a 9-fold increase of the saturation catalytic current density (jm) for Sulfite oxidation reaching 24.4 ± 0.3 μA cm–2, the highest value among reported DET-based hSO bioelectrodes. The increased electron transfer rate plays a dominating role in the enhancement of direct enzymatic current because of the improved electric contact of hSO with the electrode. The optimized hSO bioelectrode shows...
-
Transient Catalytic Voltammetry of Sulfite Oxidase Reveals Rate Limiting Conformational Changes
Journal of the American Chemical Society, 2017Co-Authors: Ting Zeng, Silke Leimkühler, Ulla Wollenberger, Vincent FourmondAbstract:Sulfite Oxidases are metalloenzymes that oxidize Sulfite to sulfate at a molybdenum active site. In vertebrate Sulfite Oxidases, the electrons generated at the Mo center are transferred to an external electron acceptor via a heme domain, which can adopt two conformations: a " closed " conformation, suitable for internal electron transfer, and an " open " conformation suitable for intermolecular electron transfer. This conformational change is an integral part of the catalytic cycle. Sulfite Oxidases have been wired to electrode surfaces, but their immobilization lead to a significant decrease in their catalytic activity, raising the question of the occurrence of the conformational change when the enzyme is on an electrode. We recorded and quantitatively modelled for the first time the transient response of the catalytic cycle of human Sulfite Oxidase immobilized on an electrode. We show that conformational changes still occur on the 1 electrode, but at a lower rate than in solution, which is the reason for the decrease in activity of Sulfite Oxidases upon immobilization.
-
effective electrochemistry of human Sulfite Oxidase immobilized on quantum dots modified indium tin oxide electrode
ACS Applied Materials & Interfaces, 2015Co-Authors: Ting Zeng, Silke Leimkühler, Joachim Koetz, Ulla WollenbergerAbstract:The bioelectrocatalytic Sulfite oxidation by human Sulfite Oxidase (hSO) on indium tin oxide (ITO) is reported, which is facilitated by functionalizing of the electrode surface with polyethylenimine (PEI)-entrapped CdS nanoparticles and enzyme. hSO was assembled onto the electrode with a high surface loading of electroactive enzyme. In the presence of Sulfite but without additional mediators, a high bioelectrocatalytic current was generated. Reference experiments with only PEI showed direct electron transfer and catalytic activity of hSO, but these were less pronounced. The application of the polyelectrolyte-entrapped quantum dots (QDs) on ITO electrodes provides a compatible surface for enzyme binding with promotion of electron transfer. Variations of the buffer solution conditions, e.g., ionic strength, pH, viscosity, and the effect of oxygen, were studied in order to understand intramolecular and heterogeneous electron transfer from hSO to the electrode. The results are consistent with a model derived for the enzyme by using flash photolysis in solution and spectroelectrochemistry and molecular dynamic simulations of hSO on monolayer-modified gold electrodes. Moreover, for the first time a photoelectrochemical electrode involving immobilized hSO is demonstrated where photoexcitation of the CdS/hSO-modified electrode lead to an enhanced generation of bioelectrocatalytic currents upon Sulfite addition. Oxidation starts already at the redox potential of the electron transfer domain of hSO and is greatly increased by application of a small overpotential to the CdS/hSO-modified ITO.
-
the electrically wired molybdenum domain of human Sulfite Oxidase is bioelectrocatalytically active
European Journal of Inorganic Chemistry, 2015Co-Authors: Roberto Spricigo, Frieder W. Scheller, Lo Gorton, Silke Leimkühler, Ulla WollenbergerAbstract:We report electron transfer between the catalytic molybdenum cofactor (Moco) domain of human Sulfite Oxidase (hSO) and electrodes through a poly(vinylpyridine)-bound [osmium(N,N'-methyl-2,2'-biimidazole)(3)](2+/3+) complex as the electron-transfer mediator. The biocatalyst was immobilized in this low-potential redox polymer on a carbon electrode. Upon the addition of Sulfite to the immobilized separate Moco domain, the generation of a significant catalytic current demonstrated that the catalytic center is effectively wired and active. The bioelectrocatalytic current of the wired separate catalytic domain reached 25% of the signal of the wired full molybdoheme enzyme hSO, in which the heme b(5) is involved in the electron-transfer pathway. This is the first report on a catalytically active wired molybdenum cofactor domain. The formal potential of this electrochemical mediator is between the potentials of the two cofactors of hSO, and as hSO can occupy several conformations in the polymer matrix, it is imaginable that electron transfer from the catalytic site to the electrode through the osmium center occurs for the hSO molecules in which the Moco domain is sufficiently accessible. The observation of catalytic oxidation currents at low potentials is favorable for applications in bioelectronic devices.
-
human Sulfite Oxidase electrochemistry on gold nanoparticles modified electrode
Bioelectrochemistry, 2012Co-Authors: Stefano Frasca, Silke Leimkühler, Oscar Rojas, Johannes Salewski, Bettina Neumann, Konstanze Stiba, Inez M Weidinger, Brigitte Tiersch, Joachim Koetz, Ulla WollenbergerAbstract:Abstract The present study reports a facile approach for Sulfite biosensing, based on enhanced direct electron transfer of a human Sulfite Oxidase ( hSO ) immobilized on a gold nanoparticles modified electrode. The spherical core shell AuNPs were prepared via a new method by reduction of HAuCl 4 with branched poly(ethyleneimine) in an ionic liquids resulting particles with a diameter less than 10 nm. These nanoparticles were covalently attached to a mercaptoundecanoic acid modified Au-electrode where then hSO was adsorbed and an enhanced interfacial electron transfer and electrocatalysis was achieved. UV/Vis and resonance Raman spectroscopy, in combination with direct protein voltammetry, are employed for the characterization of the system and reveal no perturbation of the structural integrity of the redox protein. The proposed biosensor exhibited a quick steady-state current response, within 2 s, a linear detection range between 0.5 and 5.4 μM with a high sensitivity (1.85 nA μM − 1 ). The investigated system provides remarkable advantages in the possibility to work at low applied potential and at very high ionic strength. Therefore these properties could make the proposed system useful in the development of bioelectronic devices and its application in real samples.
Graham N George - One of the best experts on this subject based on the ideXlab platform.
-
The structures of the C185S and C185A mutants of Sulfite Oxidase reveal rearrangement of the active site.
Biochemistry, 2010Co-Authors: James A Qiu, Graham N George, Heather L Wilson, Caroline Kisker, M. Jake Pushie, K. V. RajagopalanAbstract:Sulfite Oxidase (SO) catalyzes the physiologically critical conversion of Sulfite to sulfate. Enzymatic activity is dependent on the presence of the metal molybdenum complexed with a pyranopterin-dithiolene cofactor termed molybdopterin. Comparison of the amino acid sequences of SOs from a variety of sources has identified a single conserved Cys residue essential for catalytic activity. The crystal structure of chicken liver Sulfite Oxidase indicated that this residue, Cys185 in chicken SO, coordinates the Mo atom in the active site. To improve our understanding of the role of this residue in the catalytic mechanism of Sulfite Oxidase, serine and alanine variants at position 185 of recombinant chicken SO were generated. Spectroscopic and kinetic studies indicate that neither variant is capable of Sulfite oxidation. The crystal structure of the C185S variant was determined to 1.9 A resolution and to 2.4 A resolution in the presence of Sulfite, and the C185A variant to 2.8 A resolution. The structures of th...
-
active site dynamics and large scale domain motions of Sulfite Oxidase a molecular dynamics study
Journal of Physical Chemistry B, 2010Co-Authors: Jake M Pushie, Graham N GeorgeAbstract:The physiologically vital enzyme Sulfite Oxidase employs rapid intramolecular electron transfer between a molybdenum ion in the C-terminal domain (the site of Sulfite oxidation) and a heme moeity in the N-terminal domain to complete its catalytic cycle. Crystal structures of the enzyme show C- and N-terminal domain orientations that are not consistent with rapid intramolecular electron transfer. Domain motion has been postulated to explain this discrepancy. In the present work we employ molecular dynamics simulations to understand the large-scale domain motions of the enzyme. We observe motion of the N-terminal domain into an orientation similar to that postulated for rapid electron transfer. Our simulations also probe the dynamics of the active site and surrounding residues, adding a further level of structural and thermodynamic detail in understanding Sulfite Oxidase function.
-
modified active site coordination in a clinical mutant of Sulfite Oxidase
Journal of the American Chemical Society, 2007Co-Authors: Christian J Doonan, Robert M. Garrett, K. V. Rajagopalan, Heather L Wilson, Brian Bennett, Roger C Prince, Graham N GeorgeAbstract:The molybdenum site of the Arginine 160 {yields} Glutamine clinical mutant of the physiologically vital enzyme Sulfite Oxidase has been investigated by a combination of X-ray absorption spectroscopy and density functional theory calculations. We conclude that the mutant enzyme has a six-coordinate pseudo-octahedral active site with coordination of Glutamine O{sup {epsilon}} to molybdenum. This contrasts with the wild-type enzyme which is five-coordinate with approximately square-based pyramidal geometry. This difference in the structure of the molybdenum site explains many of the properties of the mutant enzyme which have previously been reported.
-
High-Resolution EXAFS of the Active Site of Human Sulfite Oxidase: Comparison With Density Functional Theory And X-Ray Crystallographic Results
Inorganic Chemistry, 2006Co-Authors: Hugh H Harris, Graham N GeorgeAbstract:Much of our knowledge about molybdenum enzymes has originated from EXAFS spectroscopy. This technique provides excellent bond-length accuracy but has only limited bond-length resolution. We have used EXAFS spectroscopy with an extended data range in an attempt to improve bond-length resolution for the molybdenum enzyme Sulfite Oxidase. The Mo site of Sulfite Oxidase has two oxygen and three Mo-S ligands (two from cofactor dithiolene plus a cysteine). For the oxidized (MO VI ) enzyme, we find that the three Mo-S bond lengths are very similar (within 0.05 A) at 2.41 A, as are the Mo=O ligands at 1.72 A. Density functional theory shows that this is consistent with the proposed active-site structure. The reduced (MO IV ) enzyme shows two Mo-S bond lengths at 2.35 A A and one at 2.41 A (assigned to cofactor dithiolene and cysteine, respectively, from DFT), together with one Mo=O at 1.72 A and one Mo-OH 2 at 2.30 A.
-
the molybdenum site of Sulfite Oxidase a comparison of wild type and the cysteine 207 to serine mutant using x ray absorption spectroscopy
Journal of the American Chemical Society, 1996Co-Authors: Graham N George, Robert M. Garrett, Roger C Prince, K. V. RajagopalanAbstract:X-ray absorption spectroscopy at the molybdenum and sulfur K-edges has been used to probe the active site of wild-type and cysteine 207 → serine mutant human Sulfite Oxidases. We compare the active site structures in the Mo(VI) oxidation states: the wild-type enzyme possesses two MoO ligands at 1.71 A and three Mo−S ligands at 2.41 A. The mutant molybdenum site is a novel trioxo site with MoO bond lengths of 1.74 A, with two Mo−S ligands at 2.47 A. We conclude that cysteine 207 is a ligand of molybdenum in wild-type human Sulfite Oxidase, and that, in the mutant, the Mo is ligated to an extra oxo group rather than the hydroxyl of the substituent serine 207.
Robert M. Garrett - One of the best experts on this subject based on the ideXlab platform.
-
modified active site coordination in a clinical mutant of Sulfite Oxidase
Journal of the American Chemical Society, 2007Co-Authors: Christian J Doonan, Robert M. Garrett, K. V. Rajagopalan, Heather L Wilson, Brian Bennett, Roger C Prince, Graham N GeorgeAbstract:The molybdenum site of the Arginine 160 {yields} Glutamine clinical mutant of the physiologically vital enzyme Sulfite Oxidase has been investigated by a combination of X-ray absorption spectroscopy and density functional theory calculations. We conclude that the mutant enzyme has a six-coordinate pseudo-octahedral active site with coordination of Glutamine O{sup {epsilon}} to molybdenum. This contrasts with the wild-type enzyme which is five-coordinate with approximately square-based pyramidal geometry. This difference in the structure of the molybdenum site explains many of the properties of the mutant enzyme which have previously been reported.
-
isolated Sulfite Oxidase deficiency identification of 12 novel suox mutations in 10 patients
Human Mutation, 2002Co-Authors: Jean L. Johnson, Robert M. Garrett, Caroline Kisker, Katharine E Coyne, Marietherese Zabot, Claude Dorche, K. V. RajagopalanAbstract:We report twelve novel mutations in patients with isolated Sulfite Oxidase deficiency. Themutations are in SUOX, the gene that encodes the molybdohemoprotein Sulfite Oxidase.These include two frameshift mutations, a four-basepair deletion (562del4) and a single-basepair insertion (113insC), both resulting in premature termination. Nonsense mutationspredicting Y343X and Q364X substitutions were identified in a homozygous state in threepatients, the latter in two sibs. The remaining eight are missense mutations generating singleamino acid substitutions. From the position of the substituted residues, seven of thesemutations are considered to be causative of the enzyme deficiency: I201L, R211Q, G305S,R309H, K322R, Q339R, and W393R. The eighth, a C>T transition, predicts an R319Csubstitution, which could affect the binding of the molybdenum cofactor and thus severelyreduce Sulfite Oxidase activity. This mutation, however, is downstream of a frameshiftmutation and is therefore not the causative mutation in this individual.
-
human Sulfite Oxidase r160q identification of the mutation in a Sulfite Oxidase deficient patient and expression and characterization of the mutant enzyme
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Robert M. Garrett, Jean L. Johnson, Tyler N Graf, Annette Feigenbaum, K. V. RajagopalanAbstract:Sulfite Oxidase catalyzes the terminal reaction in the degradation of sulfur amino acids. Genetic deficiency of Sulfite Oxidase results in neurological abnormalities and often leads to death at an early age. The mutation in the Sulfite Oxidase gene responsible for Sulfite Oxidase deficiency in a 5-year-old girl was identified by sequence analysis of cDNA obtained from fibroblast mRNA to be a guanine to adenine transition at nucleotide 479 resulting in the amino acid substitution of Arg-160 to Gln. Recombinant protein containing the R160Q mutation was expressed in Escherichia coli, purified, and characterized. The mutant protein contained its full complement of molybdenum and heme, but exhibited 2% of native activity under standard assay conditions. Absorption spectroscopy of the isolated molybdenum domains of native Sulfite Oxidase and of the R160Q mutant showed significant differences in the 480- and 350-nm absorption bands, suggestive of altered geometry at the molybdenum center. Kinetic analysis of the R160Q protein showed an increase in Km for Sulfite combined with a decrease in kcat resulting in a decrease of nearly 1,000-fold in the apparent second-order rate constant kcat/Km. Kinetic parameters for the in vitro generated R160K mutant were found to be intermediate in value between those of the native protein and the R160Q mutant. Native Sulfite Oxidase was rapidly inactivated by phenylglyoxal, yielding a modified protein with kinetic parameters mimicking those of the R160Q mutant. It is proposed that Arg-160 attracts the anionic substrate Sulfite to the binding site near the molybdenum.
-
Molecular Basis of Sulfite Oxidase Deficiency from the Structure of Sulfite Oxidase
Cell, 1997Co-Authors: Caroline Kisker, John H. Enemark, Robert M. Garrett, K. V. Rajagopalan, Hermann Schindelin, Andrew Pacheco, William A Wehbi, Douglas C. ReesAbstract:Abstract The molybdenum-containing enzyme Sulfite Oxidase catalyzes the conversion of Sulfite to sulfate, the terminal step in the oxidative degradation of cysteine and methionine. Deficiency of this enzyme in humans usually leads to major neurological abnormalities and early death. The crystal structure of chicken liver Sulfite Oxidase at 1.9 A resolution reveals that each monomer of the dimeric enzyme consists of three domains. At the active site, the Mo is penta-coordinated by three sulfur ligands, one oxo group, and one water/hydroxo. A sulfate molecule adjacent to the Mo identifies the substrate binding pocket. Four variants associated with Sulfite Oxidase deficiency have been identified: two mutations are near the sulfate binding site, while the other mutations occur within the domain mediating dimerization.
-
the molybdenum site of Sulfite Oxidase a comparison of wild type and the cysteine 207 to serine mutant using x ray absorption spectroscopy
Journal of the American Chemical Society, 1996Co-Authors: Graham N George, Robert M. Garrett, Roger C Prince, K. V. RajagopalanAbstract:X-ray absorption spectroscopy at the molybdenum and sulfur K-edges has been used to probe the active site of wild-type and cysteine 207 → serine mutant human Sulfite Oxidases. We compare the active site structures in the Mo(VI) oxidation states: the wild-type enzyme possesses two MoO ligands at 1.71 A and three Mo−S ligands at 2.41 A. The mutant molybdenum site is a novel trioxo site with MoO bond lengths of 1.74 A, with two Mo−S ligands at 2.47 A. We conclude that cysteine 207 is a ligand of molybdenum in wild-type human Sulfite Oxidase, and that, in the mutant, the Mo is ligated to an extra oxo group rather than the hydroxyl of the substituent serine 207.
