A5009567046- Porphyrin Metabolism and Disorders
- Gout, Hyperuricemia, Uric Acid
- Biochemical and Molecular Research
- Metabolism and Genetic Disorders
- Photoreceptor and optogenetics research
- Enzyme Structure and Function
- Folate and B Vitamins Research
- Polyamine Metabolism and Applications
- Synthesis and Characterization of Heterocyclic Compounds
- RNA and protein synthesis mechanisms
- Receptor Mechanisms and Signaling
- Lipid Membrane Structure and Behavior
- Peptidase Inhibition and Analysis
- Biochemical effects in animals
- Metalloenzymes and iron-sulfur proteins
- Food Industry and Aquatic Biology
- Synthesis of Organic Compounds
- Bacterial Genetics and Biotechnology
- DNA and Nucleic Acid Chemistry
- Analytical Chemistry and Chromatography
- Carcinogens and Genotoxicity Assessment
- Neuroscience and Neuropharmacology Research
- Heme Oxygenase-1 and Carbon Monoxide
- Retinal Development and Disorders
- Biochemical and Structural Characterization
University of Toronto
2008-2021
Canada Research Chairs
2000-2021
Princess Margaret Cancer Centre
2000-2011
Ontario Institute for Cancer Research
2008-2011
University Health Network
2003-2010
Nippon Medical School
2000-2010
Cancer Research Institute
2010
Pennsylvania State University
1991-1992
Mammalian xanthine oxidoreductases, which catalyze the last two steps in formation of urate, are synthesized as dehydrogenase form (XDH) but can be readily converted to oxidase (XO) by oxidation sulfhydryl residues or proteolysis. Here, we present crystal structure dimeric ( M r , 290,000) bovine milk XDH at 2.1-Å resolution and XO 2.5-Å describe major changes that occur on proteolytic transformation form. Each molecule is composed an N-terminal 20-kDa domain containing iron sulfur centers,...
Molybdenum is widely distributed in biology and usually found as a mononuclear metal center the active sites of many enzymes catalyzing oxygen atom transfer. The molybdenum hydroxylases are distinct from other biological systems hydroxylation reactions that incorporated into product derived water rather than molecular oxygen. Here, we present crystal structure key intermediate reaction xanthine oxidoreductase with slow substrate, which carbon–oxygen bond formed, yet remains complexed to...
Mammalian xanthine dehydrogenase can be converted to oxidase by modification of cysteine residues or proteolysis the enzyme polypeptide chain. Here we present evidence that Cys535 and Cys992 rat liver are indeed involved in rapid conversion from oxidase. The purified mutants C535A and/or C992R were significantly resistant incubation with 4,4′-dithiodipyridine, whereas recombinant wild-type readily type, indicating these responsible for conversion. C535A/C992R mutant, however, very slowly...
Inhibitors of xanthine oxidoreductase block conversion to uric acid and are therefore potentially useful for treatment hyperuricemia or gout. We determined the crystal structure reduced bovine milk complexed with oxipurinol at 2.0 A resolution. Clear electron density was observed between N2 nitrogen molybdenum atom molybdopterin cofactor, indicating that coordinated directly molybdenum. Oxipurinol forms hydrogen bonds glutamate 802, arginine 880, 1261, which have previously been shown be...
The advent of ultrafast highly brilliant coherent X-ray free-electron laser sources has driven the development novel structure-determination approaches for proteins, and promises visualization protein dynamics on sub-picosecond timescales with full atomic resolution. Significant efforts are being applied to sample-delivery systems that allow these unique be most efficiently exploited high-throughput serial femtosecond crystallography. Here, next iteration a fixed-target crystallography chip...
Y-700 (1-[3-Cyano-4-(2,2-dimethylpropoxy)phenyl]-1<i>H</i>-pyrazole-4-carboxylic acid) is a newly synthesized inhibitor of xanthine oxidoreductase (XOR). Steady-state kinetics with the bovine milk enzyme indicated mixed type inhibition <i>K</i><sub>i</sub> and ′ values 0.6 3.2 nM, respectively. Titration experiments showed that bound tightly both to active sulfo-form inactive desulfo-form <i>K</i><sub>d</sub> 0.9 2.8 X-ray crystallographic analysis enzyme-inhibitor complex revealed closely...
In mammals, xanthine oxidoreductase is synthesized as a dehydrogenase (XDH) but can be readily converted to its oxidase form (XO) either by proteolysis or modification of cysteine residues. The crystal structures bovine milk XDH and XO demonstrated that atoms in the highly charged active-site loop (Gln-423-Lys-433) around FAD cofactor underwent large dislocations during conversion, blocking approach NAD+ substrate well changing electrostatic environment FAD. Here we identify unique cluster...
The problem of antibiotic resistance has prompted the search for new antibiotics with novel mechanisms action. Analogues A54556 cyclic acyldepsipeptides (ADEPs) represent an attractive class antimicrobial agents that act through dysregulation caseinolytic protease (ClpP). Previous studies have shown ADEPs are active against Gram-positive bacteria (e.g., MRSA, VRE, PRSP (penicillin-resistant Streptococcus pneumoniae)); however, there currently few examining Gram-negative bacteria. In this...
For the two proteins myoglobin and fluoroacetate dehalogenase, we present a systematic comparison of crystallographic diffraction data collected by serial femtosecond (SFX) synchrotron crystallography (SSX). To maximize comparability, used same batch micron-sized crystals, sample delivery device, analysis software. Overall figures merit indicate that both radiation sources are equivalent quality. proteins, reasonable statistics can be obtained with approximately 5000 room-temperature images...
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMechanism of DNA replication fidelity for three mutants polymerase I: Klenow fragment KF(exo+), KF(polA5), and KF(exo-)Bryan T. Eger, Robert D. Kuchta, Steven S. Carroll, Patricia A. Benkovic, Michael E. Dahlberg, Catherine M. Joyce, Stephen J. BenkovicCite this: Biochemistry 1991, 30, 5, 1441–1448Publication Date (Print):February 1991Publication History Published online1 May 2002Published inissue 5 February...
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThe minimal kinetic mechanism for misincorporation by DNA polymerase I (Klenow fragment)Bryan T. Eger and Stephen J. BenkovicCite this: Biochemistry 1992, 31, 38, 9227–9236Publication Date (Print):September 1, 1992Publication History Published online1 May 2002Published inissue 1 September 1992https://pubs.acs.org/doi/10.1021/bi00153a016https://doi.org/10.1021/bi00153a016research-articleACS PublicationsRequest reuse permissionsArticle...
In mammals, xanthine oxidoreductase can exist as dehydrogenase (XDH) and oxidase (XO). The two enzymes possess common redox active cofactors, which form an electron transfer (ET) pathway terminated by a flavin cofactor. spite of identical protein primary structures, the potential difference between XDH XO for semiquinone/hydroquinone pair (E(sq/hq)) is ~170 mV, striking difference. former greatly prefers NAD(+) ultimate substrate ET from iron-sulfur cluster FeS-II via while latter only...
Mammalian xanthine oxidoreductase can exist in both dehydrogenase and oxidase forms. Conversion between the two is implicated such diverse processes as lactation, anti‐bacterial activity, reperfusion injury a growing number of diseases. We have constructed variant rat liver enzyme that lacks carboxy‐terminal amino acids 1316–1331; it appears to assume an intermediate form, exhibiting mixture activities. The purified protein retained ~ 50–70% activity even after prolonged dithiothreitol...
Two contradictory models have been proposed for the binding mode of substrate xanthine to and its activation mechanism by oxidoreductase. In an effort distinguish between two models, we determined crystal structures urate complexes demolybdo-form D428A mutant rat oxidoreductase at 1.7 Å reduced bovine milk enzyme 2.1 Å, latter representing a reaction intermediate. The results clearly indicate catalytically relevant xanthine.
Microbial rhodopsins are versatile and ubiquitous retinal-binding proteins that function as light-driven ion pumps, light-gated channels, photosensors, with potential utility optogenetic tools for altering membrane in target cells. Insights from crystal structures have been central understanding proton, sodium, chloride transport mechanisms of microbial rhodopsins. Two three known groups anion the archaeal halorhodopsins (HRs) bacterial chloride-pumping rhodopsins, structurally...
In order to probe the structural basis of stereoselectivity in serine protease family, a series enantiomeric boronic acids RCH2CH(NHCOCH3)B(OH)2 has been synthesized and kinetically characterized as transition-state analog inhibitors using α-chymotrypsin subtilisin Carlsberg model systems. When R-substituent this was changed from p-chlorophenyl 1-naphthyl group, α-chymotrypsin, but not subtilisin, reversed its usual preference for l-enantiomers bound more tightly d-enantiomer [Martichonok,...
Abstract Bacterial ClpP is a highly conserved, cylindrical, self-compartmentalizing serine protease required for maintaining cellular proteostasis. Small molecule acyldepsipeptides (ADEPs) and activators of self-compartmentalized proteases 1 (ACP1s) cause dysregulation activation ClpP, leading to bacterial cell death, highlighting their potential use as novel antibiotics. Structural changes in Neisseria meningitidis Escherichia coli upon binding ACP1 ADEP analogs were probed by X-ray...
Xanthine dehydrogenase catalyzes the oxidation of hypoxanthine to xanthine and further uric acid. The enzyme is target anti-gout drug allopurinol its involvement in postischemic reperfusion injury presently being defined. Each subunit homodimeric 290 kDa contains four cofactors: one Mo-pterin, two [2Fe–2S] clusters FAD. Both (XDH) proteolytically modified oxidase form (XO) from bovine milk have been crystallized. XO crystals belong space group C2221, with unit-cell parameters a = 116.3, b...