Publications by authors named "Gunes Bender"

12 Publications

  • Page 1 of 1

Membrane-dependent Activities of Human 15-LOX-2 and Its Murine Counterpart: IMPLICATIONS FOR MURINE MODELS OF ATHEROSCLEROSIS.

J Biol Chem 2016 09 19;291(37):19413-24. Epub 2016 Jul 19.

From the Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803 and

The enzyme encoded by the ALOX15B gene has been linked to the development of atherosclerotic plaques in humans and in a mouse model of hypercholesterolemia. In vitro, these enzymes, which share 78% sequence identity, generate distinct products from their substrate arachidonic acid: the human enzyme, a 15-S-hydroperoxy product; and the murine enzyme, an 8-S-product. We probed the activities of these enzymes with nanodiscs as membrane mimics to determine whether they can access substrate esterified in a bilayer and characterized their activities at the membrane interface. We observed that both enzymes transform phospholipid-esterified arachidonic acid to a 15-S-product. Moreover, when expressed in transfected HEK cells, both enzymes result in significant increases in the amounts of 15-hydroxyderivatives of eicosanoids detected. In addition, we show that 15-LOX-2 is distributed at the plasma membrane when the HEK293 cells are stimulated by the addition Ca(2+) ionophore and that cellular localization is dependent upon the presence of a putative membrane insertion loop. We also report that sequence differences between the human and mouse enzymes in this loop appear to confer distinct mechanisms of enzyme-membrane interaction for the homologues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M116.741454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016680PMC
September 2016

Crystal structure of a lipoxygenase in complex with substrate: the arachidonic acid-binding site of 8R-lipoxygenase.

J Biol Chem 2014 Nov 17;289(46):31905-31913. Epub 2014 Sep 17.

Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803,. Electronic address:

Lipoxygenases (LOX) play critical roles in mammalian biology in the generation of potent lipid mediators of the inflammatory response; consequently, they are targets for the development of isoform-specific inhibitors. The regio- and stereo-specificity of the oxygenation of polyunsaturated fatty acids by the enzymes is understood in terms of the chemistry, but structural observation of the enzyme-substrate interactions is lacking. Although several LOX crystal structures are available, heretofore the rapid oxygenation of bound substrate has precluded capture of the enzyme-substrate complex, leaving a gap between chemical and structural insights. In this report, we describe the 2.0 Å resolution structure of 8R-LOX in complex with arachidonic acid obtained under anaerobic conditions. Subtle rearrangements, primarily in the side chains of three amino acids, allow binding of arachidonic acid in a catalytically competent conformation. Accompanying experimental work supports a model in which both substrate tethering and cavity depth contribute to positioning the appropriate carbon at the catalytic machinery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M114.599662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231669PMC
November 2014

Transient B12-dependent methyltransferase complexes revealed by small-angle X-ray scattering.

J Am Chem Soc 2012 Oct 19;134(43):17945-54. Epub 2012 Oct 19.

Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

In the Wood-Ljungdahl carbon fixation pathway, protein-protein interactions between methyltransferase (MeTr) and corrinoid iron-sulfur protein (CFeSP) are required for the transfer of a methyl group. While crystal structures have been determined for MeTr and CFeSP both free and in complex, solution structures have not been established. Here, we examine the transient interactions between MeTr and CFeSP in solution using anaerobic small-angle X-ray scattering (SAXS) and present a global analysis approach for the deconvolution of heterogeneous mixtures formed by weakly interacting proteins. We further support this SAXS analysis with complementary results obtained by anaerobic isothermal titration calorimetry. Our results indicate that solution conditions affect the cooperativity with which CFeSP binds to MeTr, resulting in two distinct CFeSP/MeTr complexes with differing oligomeric compositions, both of which are active. One assembly resembles the CFeSP/MeTr complex observed crystallographically with 2:1 protein stoichiometry, while the other best fits a 1:1 CFeSP/MeTr arrangement. These results demonstrate the value of SAXS in uncovering the rich solution behavior of transient protein interactions visualized by crystallography.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ja3055782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3484714PMC
October 2012

Redox, haem and CO in enzymatic catalysis and regulation.

Biochem Soc Trans 2012 Jun;40(3):501-7

Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, U.S.A.

The present paper describes general principles of redox catalysis and redox regulation in two diverse systems. The first is microbial metabolism of CO by the Wood-Ljungdahl pathway, which involves the conversion of CO or H2/CO2 into acetyl-CoA, which then serves as a source of ATP and cell carbon. The focus is on two enzymes that make and utilize CO, CODH (carbon monoxide dehydrogenase) and ACS (acetyl-CoA synthase). In this pathway, CODH converts CO2 into CO and ACS generates acetyl-CoA in a reaction involving Ni·CO, methyl-Ni and acetyl-Ni as catalytic intermediates. A 70 Å (1 Å=0.1 nm) channel guides CO, generated at the active site of CODH, to a CO 'cage' near the ACS active site to sequester this reactive species and assure its rapid availability to participate in a kinetically coupled reaction with an unstable Ni(I) state that was recently trapped by photolytic, rapid kinetic and spectroscopic studies. The present paper also describes studies of two haem-regulated systems that involve a principle of metabolic regulation interlinking redox, haem and CO. Recent studies with HO2 (haem oxygenase-2), a K+ ion channel (the BK channel) and a nuclear receptor (Rev-Erb) demonstrate that this mode of regulation involves a thiol-disulfide redox switch that regulates haem binding and that gas signalling molecules (CO and NO) modulate the effect of haem.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BST20120083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3553215PMC
June 2012

Visualizing molecular juggling within a B12-dependent methyltransferase complex.

Nature 2012 Mar 14;484(7393):265-9. Epub 2012 Mar 14.

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Derivatives of vitamin B(12) are used in methyl group transfer in biological processes as diverse as methionine synthesis in humans and CO(2) fixation in acetogenic bacteria. This seemingly straightforward reaction requires large, multimodular enzyme complexes that adopt multiple conformations to alternately activate, protect and perform catalysis on the reactive B(12) cofactor. Crystal structures determined thus far have provided structural information for only fragments of these complexes, inspiring speculation about the overall protein assembly and conformational movements inherent to activity. Here we present X-ray crystal structures of a complete 220 kDa complex that contains all enzymes responsible for B(12)-dependent methyl transfer, namely the corrinoid iron-sulphur protein and its methyltransferase from the model acetogen Moorella thermoacetica. These structures provide the first three-dimensional depiction of all protein modules required for the activation, protection and catalytic steps of B(12)-dependent methyl transfer. In addition, the structures capture B(12) at multiple locations between its 'resting' and catalytic positions, allowing visualization of the dramatic protein rearrangements that enable methyl transfer and identification of the trajectory for B(12) movement within the large enzyme scaffold. The structures are also presented alongside in crystallo spectroscopic data, which confirm enzymatic activity within crystals and demonstrate the largest known conformational movements of proteins in a crystalline state. Taken together, this work provides a model for the molecular juggling that accompanies turnover and helps explain why such an elaborate protein framework is required for such a simple, yet biologically essential reaction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature10916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326194PMC
March 2012

Metal centers in the anaerobic microbial metabolism of CO and CO2.

Metallomics 2011 Aug 6;3(8):797-815. Epub 2011 Jun 6.

Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.

Carbon dioxide and carbon monoxide are important components of the carbon cycle. Major research efforts are underway to develop better technologies to utilize the abundant greenhouse gas, CO(2), for harnessing 'green' energy and producing biofuels. One strategy is to convert CO(2) into CO, which has been valued for many years as a synthetic feedstock for major industrial processes. Living organisms are masters of CO(2) and CO chemistry and, here, we review the elegant ways that metalloenzymes catalyze reactions involving these simple compounds. After describing the chemical and physical properties of CO and CO(2), we shift focus to the enzymes and the metal clusters in their active sites that catalyze transformations of these two molecules. We cover how the metal centers on CO dehydrogenase catalyze the interconversion of CO and CO(2) and how pyruvate oxidoreductase, which contains thiamin pyrophosphate and multiple Fe(4)S(4) clusters, catalyzes the addition and elimination of CO(2) during intermediary metabolism. We also describe how the nickel center at the active site of acetyl-CoA synthase utilizes CO to generate the central metabolite, acetyl-CoA, as part of the Wood-Ljungdahl pathway, and how CO is channelled from the CO dehydrogenase to the acetyl-CoA synthase active site. We cover how the corrinoid iron-sulfur protein interacts with acetyl-CoA synthase. This protein uses vitamin B(12) and a Fe(4)S(4) cluster to catalyze a key methyltransferase reaction involving an organometallic methyl-Co(3+) intermediate. Studies of CO and CO(2) enzymology are of practical significance, and offer fundamental insights into important biochemical reactions involving metallocenters that act as nucleophiles to form organometallic intermediates and catalyze C-C and C-S bond formations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c1mt00042jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3964926PMC
August 2011

Evidence that ferredoxin interfaces with an internal redox shuttle in Acetyl-CoA synthase during reductive activation and catalysis.

Biochemistry 2011 Jan 21;50(2):276-86. Epub 2010 Dec 21.

Department of Biological Chemistry, University of Michigan, Ann Arbor, 48109, United States.

Acetyl-CoA synthase (ACS), a subunit of the bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex of Moorella thermoacetica requires reductive activation in order to catalyze acetyl-CoA synthesis and related partial reactions, including the CO/[1-(14)C]-acetyl-CoA exchange reaction. We show that the M. thermoacetica ferredoxin(II) (Fd-II), which harbors two [4Fe-4S] clusters and is an electron acceptor for CODH, serves as a redox activator of ACS. The level of activation depends on the oxidation states of both ACS and Fd-II, which strongly suggests that Fd-II acts as a reducing agent. By the use of controlled potential enzymology, the midpoint reduction potential for the catalytic one-electron redox-active species in the CO/acetyl-CoA exchange reaction is -511 mV, which is similar to the midpoint reduction potential that was earlier measured for other reactions involving ACS. Incubation of ACS with Fd-II and CO leads to the formation of the NiFeC species, which also supports the role of Fd-II as a reductant for ACS. In addition to being a reductant, Fd-II can accept electrons from acetylated ACS, as observed by the increased intensity of the EPR spectrum of reduced Fd-II, indicating that there is a stored electron within an "electron shuttle" in the acetyl-Ni(II) form of ACS. This "shuttle" is proposed to serve as a redox mediator during activation and at different steps of the ACS catalytic cycle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi101511rDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3077469PMC
January 2011

Infrared and EPR spectroscopic characterization of a Ni(I) species formed by photolysis of a catalytically competent Ni(I)-CO intermediate in the acetyl-CoA synthase reaction.

Biochemistry 2010 Sep;49(35):7516-23

Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.

Acetyl-CoA synthase (ACS) catalyzes the synthesis of acetyl-CoA from CO, coenzyme A (CoA), and a methyl group from the CH(3)-Co(3+) site in the corrinoid iron-sulfur protein (CFeSP). These are the key steps in the Wood-Ljungdahl pathway of anaerobic CO and CO(2) fixation. The active site of ACS is the A-cluster, which is an unusual nickel-iron-sulfur cluster. There is significant evidence for the catalytic intermediacy of a CO-bound paramagnetic Ni species, with an electronic configuration of [Fe(4)S(4)](2+)-(Ni(p)(+)-CO)-(Ni(d)(2+)), where Ni(p) and Ni(d) represent the Ni centers in the A-cluster that are proximal and distal to the [Fe(4)S(4)](2+) cluster, respectively. This well-characterized Ni(p)(+)-CO intermediate is often called the NiFeC species. Photolysis of the Ni(p)(+)-CO state generates a novel Ni(p)(+) species (A(red)*) with a rhombic electron paramagnetic resonance spectrum (g values of 2.56, 2.10, and 2.01) and an extremely low (1 kJ/mol) barrier for recombination with CO. We suggest that the photolytically generated A(red)* species is (or is similar to) the Ni(p)(+) species that binds CO (to form the Ni(p)(+)-CO species) and the methyl group (to form Ni(p)-CH(3)) in the ACS catalytic mechanism. The results provide support for a binding site (an "alcove") for CO near Ni(p), indicated by X-ray crystallographic studies of the Xe-incubated enzyme. We propose that, during catalysis, a resting Ni(p)(2+) state predominates over the active Ni(p)(+) species (A(red)*) that is trapped by the coupling of a one-electron transfer step to the binding of CO, which pulls the equilibrium toward Ni(p)(+)-CO formation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi1010128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2932805PMC
September 2010

Function of Ech hydrogenase in ferredoxin-dependent, membrane-bound electron transport in Methanosarcina mazei.

J Bacteriol 2010 Feb 30;192(3):674-8. Epub 2009 Nov 30.

Institute of Microbiology and Biotechnology, University of Bonn, Meckenheimer Allee 168, 53115 Bonn, Germany.

Reduced ferredoxin is an intermediate in the methylotrophic and aceticlastic pathway of methanogenesis and donates electrons to membrane-integral proteins, which transfer electrons to the heterodisulfide reductase. A ferredoxin interaction has been observed previously for the Ech hydrogenase. Here we present a detailed analysis of a Methanosarcina mazei Delta ech mutant which shows decreased ferredoxin-dependent membrane-bound electron transport activity, a lower growth rate, and faster substrate consumption. Evidence is presented that a second protein whose identity is unknown oxidizes reduced ferredoxin, indicating an involvement in methanogenesis from methylated C(1) compounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JB.01307-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2812462PMC
February 2010

Identification of the substrate radical intermediate derived from ethanolamine during catalysis by ethanolamine ammonia-lyase.

Biochemistry 2008 Oct 1;47(43):11360-6. Epub 2008 Oct 1.

Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53726, USA.

Rapid-mix freeze-quench (RMFQ) methods and electron paramagnetic resonance (EPR) spectroscopy have been used to characterize the steady-state radical in the deamination of ethanolamine catalyzed by adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL). EPR spectra of the radical intermediates formed with the substrates, [1-13C]ethanolamine, [2-13C]ethanolamine, and unlabeled ethanolamine were acquired using RMFQ trapping methods from 10 ms to completion of the reaction. Resolved 13C hyperfine splitting in EPR spectra of samples prepared with [1-13C]ethanolamine and the absence of such splitting in spectra of samples prepared with [2-13C]ethanolamine show that the unpaired electron is localized on C1 (the carbinol carbon) of the substrate. The 13C splitting from C1 persists from 10 ms throughout the time course of substrate turnover, and there was no evidence of a detectable amount of a product like radical having unpaired spin on C2. These results correct an earlier assignment for this radical intermediate [Warncke, K., et al. (1999) J. Am. Chem. Soc. 121, 10522-10528]. The EPR signals of the substrate radical intermediate are altered by electron spin coupling to the other paramagnetic species, cob(II)alamin, in the active site. The dipole-dipole and exchange interactions as well as the 1-13C hyperfine splitting tensor were analyzed via spectral simulations. The sign of the isotropic exchange interaction indicates a weak ferromagnetic coupling of the two unpaired electrons. A Co2+-radical distance of 8.7 A was obtained from the magnitude of the dipole-dipole interaction. The orientation of the principal axes of the 13C hyperfine splitting tensor shows that the long axis of the spin-bearing p orbital on C1 of the substrate radical makes an angle of approximately 98 degrees with the unique axis of the d(z2) orbital of Co2+.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi801316vDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2631207PMC
October 2008

The contribution of neural crest cells to the nuchal bone and plastron of the turtle shell.

Integr Comp Biol 2007 Sep 1;47(3):401-8. Epub 2007 Jun 1.

*Department of Biology, Swarthmore College, 500 College Avenue, Swarthmore, PA 19081 USA; Swarthmore College, presently at Department of Chemistry, University of Wisconsin, Madison, WI 53706; Science Division, Friends Central School, 1101 City Avenue, Wynnewood, PA 19096 USA; Biology Department, Millersville University, PO Box 1002, Millersville, PA 17551 USA.

The origin of the turtle plastron is not well understood, and these nine bones have been homologized to the exoskeletal components of the clavicles, the interclavicular bone, and gastralia. Earlier data from our laboratory showed that the plastral bone-forming cells stained positively for HNK-1 and PDGFRα, two markers of skeletogenic neural crest cells. We have now shown that the HNK-1(+) cells are also positive for p75 and FoxD3, affirming their neural crest identity. These cells originate from the dorsal neural tube of stage-17 turtle embryos, several days after the original wave of neural crest cells have migrated and differentiated. Moreover, we have demonstrated the existence of a staging area, above the neural tube and vertebrae, where these late-emigrating neural crest cells collect. After residing in the carapacial staging area, these cells migrate to form the plastral bones. We also demonstrate that one bone of the carapace, the nuchal bone, also stains with HNK-1 and with antibodies to PDGFRα. The nuchal bone shares several other properties with the plastral bones, suggesting that it, too, is derived from neural crest cells. Alligator gastralia stain for HNK-1, while their ribs do not, thus suggesting that the gastralial precursor may also be derived from neural crest cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/icb/icm020DOI Listing
September 2007

How the turtle forms its shell: a paracrine hypothesis of carapace formation.

J Exp Zool B Mol Dev Evol 2005 Nov;304(6):558-69

Department of Biology, Swarthmore College, Swarthmore, Pennsylvania 19081, USA.

We propose a two-step model for the evolutionary origin of the turtle shell. We show here that the carapacial ridge (CR) is critical for the entry of the ribs into the dorsal dermis. Moreover, we demonstrate that the maintenance of the CR and its ability to attract the migrating rib precursor cells depend upon fibroblast growth factor (FGF) signaling. Inhibitors of FGF allow the CR to degenerate, with the consequent migration of ribs along the ventral body wall. Beads containing FGF10 can rearrange rib migration in the chick, suggesting that the CR FGF10 plays an important role in attracting the rib rudiments. The co-ordinated growth of the carapacial plate and the ribs may be a positive feedback loop (similar to that of the limbs) caused by the induction of Fgf8 in the distal tips of the ribs by the FGF10-secreting mesenchyme of the CR. Once in the dermis, the ribs undergo endochrondral ossification. We provide evidence that the ribs act as signaling centers for the dermal ossification and that this ossification is due to bone morphogenetic proteins secreted by the rib. Thus, once the ribs are within the dermis, the ossification of the dermis is not difficult to achieve. This relatively rapid means of carapace formation would allow for the appearance of turtles in the fossil record without obvious intermediates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jez.b.21059DOI Listing
November 2005