Publications by authors named "Marie E Fraser"

28 Publications

  • Page 1 of 1

Second distinct conformation of the phosphohistidine loop in succinyl-CoA synthetase.

Acta Crystallogr D Struct Biol 2021 Mar 19;77(Pt 3):357-368. Epub 2021 Feb 19.

Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada.

Succinyl-CoA synthetase (SCS) catalyzes a reversible reaction that is the only substrate-level phosphorylation in the citric acid cycle. One of the essential steps for the transfer of the phosphoryl group involves the movement of the phosphohistidine loop between active site I, where CoA, succinate and phosphate bind, and active site II, where the nucleotide binds. Here, the first crystal structure of SCS revealing the conformation of the phosphohistidine loop in site II of the porcine GTP-specific enzyme is presented. The phosphoryl transfer bridges a distance of 29 Å between the binding sites for phosphohistidine in site I and site II, so these crystal structures support the proposed mechanism of catalysis by SCS. In addition, a second succinate-binding site was discovered at the interface between the α- and β-subunits of SCS, and another magnesium ion was found that interacts with the side chains of Glu141β and Glu204β via water-mediated interactions. These glutamate residues interact with the active-site histidine residue when it is bound in site II.
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http://dx.doi.org/10.1107/S2059798321000334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919408PMC
March 2021

Tartryl-CoA inhibits succinyl-CoA synthetase.

Acta Crystallogr F Struct Biol Commun 2020 Jul 1;76(Pt 7):302-308. Epub 2020 Jul 1.

Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada.

Succinyl-CoA synthetase (SCS) catalyzes the only substrate-level phosphorylation step in the tricarboxylic acid cycle. Human GTP-specific SCS (GTPSCS), an αβ-heterodimer, was produced in Escherichia coli. The purified protein crystallized from a solution containing tartrate, CoA and magnesium chloride, and a crystal diffracted to 1.52 Å resolution. Tartryl-CoA was discovered to be bound to GTPSCS. The CoA portion lies in the amino-terminal domain of the α-subunit and the tartryl end extends towards the catalytic histidine residue. The terminal carboxylate binds to the phosphate-binding site of GTPSCS.
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http://dx.doi.org/10.1107/S2053230X20008201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336359PMC
July 2020

Identification of the active site residues in ATP-citrate lyase's carboxy-terminal portion.

Protein Sci 2019 10 27;28(10):1840-1849. Epub 2019 Aug 27.

Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.

ATP-citrate lyase (ACLY) catalyzes production of acetyl-CoA and oxaloacetate from CoA and citrate using ATP. In humans, this cytoplasmic enzyme connects energy metabolism from carbohydrates to the production of lipids. In certain bacteria, ACLY is used to fix carbon in the reductive tricarboxylic acid cycle. The carboxy(C)-terminal portion of ACLY shows sequence similarity to citrate synthase of the tricarboxylic acid cycle. To investigate the roles of residues of ACLY equivalent to active site residues of citrate synthase, these residues in ACLY from Chlorobium limicola were mutated, and the proteins were investigated using kinetics assays and biophysical techniques. To obtain the crystal structure of the C-terminal portion of ACLY, full-length C. limicola ACLY was cleaved, first non-specifically with chymotrypsin and subsequently with Tobacco Etch Virus protease. Crystals of the C-terminal portion diffracted to high resolution, providing structures that show the positions of active site residues and how ACLY tetramerizes.
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http://dx.doi.org/10.1002/pro.3708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739821PMC
October 2019

ATP-specificity of succinyl-CoA synthetase from Blastocystis hominis.

Acta Crystallogr D Struct Biol 2019 Jul 26;75(Pt 7):647-659. Epub 2019 Jun 26.

Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada.

Succinyl-CoA synthetase (SCS) catalyzes the only step of the tricarboxylic acid cycle that leads to substrate-level phosphorylation. Some forms of SCS are specific for ADP/ATP or for GDP/GTP, while others can bind all of these nucleotides, generally with different affinities. The theory of `gatekeeper' residues has been proposed to explain the nucleotide-specificity. Gatekeeper residues lie outside the binding site and create specific electrostatic interactions with incoming nucleotides to determine whether the nucleotides can enter the binding site. To test this theory, the crystal structure of the nucleotide-binding domain in complex with Mg-ADP was determined, as well as the structures of four proteins with single mutations, K46βE, K114βD, V113βL and L227βF, and one with two mutations, K46βE/K114βD. The crystal structures show that the enzyme is specific for ADP/ATP because of interactions between the nucleotide and the binding site. Nucleotide-specificity is provided by hydrogen-bonding interactions between the adenine base and Gln20β, Gly111β and Val113β. The O atom of the side chain of Gln20β interacts with N6 of ADP, while the side-chain N atom interacts with the carbonyl O atom of Gly111β. It is the different conformations of the backbone at Gln20β, of the side chain of Gln20β and of the linker that make the enzyme ATP-specific. This linker connects the two subdomains of the ATP-grasp fold and interacts differently with adenine and guanine bases. The mutant proteins have similar conformations, although the L227βF mutant shows structural changes that disrupt the binding site for the magnesium ion. Although the K46βE/K114βD double mutant of Blastocystis hominis SCS binds GTP better than ATP according to kinetic assays, only the complex with Mg-ADP was obtained.
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http://dx.doi.org/10.1107/S2059798319007976DOI Listing
July 2019

Binding of hydroxycitrate to human ATP-citrate lyase.

Acta Crystallogr D Struct Biol 2017 Aug 28;73(Pt 8):660-671. Epub 2017 Jul 28.

Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada.

Hydroxycitrate from the fruit of Garcinia cambogia [i.e. (2S,3S)-2-hydroxycitrate] is the best-known inhibitor of ATP-citrate lyase. Well diffracting crystals showing how the inhibitor binds to human ATP-citrate lyase were grown by modifying the protein. The protein was modified by introducing cleavage sites for Tobacco etch virus protease on either side of a disordered linker. The protein crystallized consisted of residues 2-425-ENLYFQ and S-488-810 of human ATP-citrate lyase. (2S,3S)-2-Hydroxycitrate binds in the same orientation as citrate, but the citrate-binding domain (residues 248-421) adopts a different orientation with respect to the rest of the protein (residues 4-247, 490-746 and 748-809) from that previously seen. For the first time, electron density was evident for the loop that contains His760, which is phosphorylated as part of the catalytic mechanism. The pro-S carboxylate of (2S,3S)-2-hydroxycitrate is available to accept a phosphoryl group from His760. However, when co-crystals were grown with ATP and magnesium ions as well as either the inhibitor or citrate, Mg-ADP was bound and His760 was phosphorylated. The phosphoryl group was not transferred to the organic acid. This led to the interpretation that the active site is trapped in an open conformation. The strategy of designing cleavage sites to remove disordered residues could be useful in determining the crystal structures of other proteins.
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http://dx.doi.org/10.1107/S2059798317009871DOI Listing
August 2017

Structural basis for the binding of succinate to succinyl-CoA synthetase.

Acta Crystallogr D Struct Biol 2016 08 13;72(Pt 8):912-21. Epub 2016 Jul 13.

Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada.

Succinyl-CoA synthetase catalyzes the only step in the citric acid cycle that provides substrate-level phosphorylation. Although the binding sites for the substrates CoA, phosphate, and the nucleotides ADP and ATP or GDP and GTP have been identified, the binding site for succinate has not. To determine this binding site, pig GTP-specific succinyl-CoA synthetase was crystallized in the presence of succinate, magnesium ions and CoA, and the structure of the complex was determined by X-ray crystallography to 2.2 Å resolution. Succinate binds in the carboxy-terminal domain of the β-subunit. The succinate-binding site is near both the active-site histidine residue that is phosphorylated in the reaction and the free thiol of CoA. The carboxy-terminal domain rearranges when succinate binds, burying this active site. However, succinate is not in position for transfer of the phosphoryl group from phosphohistidine. Here, it is proposed that when the active-site histidine residue has been phosphorylated by GTP, the phosphohistidine displaces phosphate and triggers the movement of the carboxylate of succinate into position to be phosphorylated. The structure shows why succinyl-CoA synthetase is specific for succinate and does not react appreciably with citrate nor with the other C4-dicarboxylic acids of the citric acid cycle, fumarate and oxaloacetate, but shows some activity with L-malate.
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http://dx.doi.org/10.1107/S2059798316010044DOI Listing
August 2016

Structural variation and uniformity among tetraloop-receptor interactions and other loop-helix interactions in RNA crystal structures.

PLoS One 2012 9;7(11):e49225. Epub 2012 Nov 9.

Department of Biological Sciences, University of Calgary, Calgary, Canada.

Tetraloop-receptor interactions are prevalent structural units in RNAs, and include the GAAA/11-nt and GNRA-minor groove interactions. In this study, we have compiled a set of 78 nonredundant loop-helix interactions from X-ray crystal structures, and examined them for the extent of their sequence and structural variation. Of the 78 interactions in the set, only four were classical GAAA/11-nt motifs, while over half (48) were GNRA-minor groove interactions. The GNRA-minor groove interactions were not a homogeneous set, but were divided into five subclasses. The most predominant subclass is characterized by two triple base pair interactions in the minor groove, flanked by two ribose zipper contacts. This geometry may be considered the "standard" GNRA-minor groove interaction, while the other four subclasses are alternative ways to form interfaces between a minor groove and tetraloop. The remaining 26 structures in the set of 78 have loops interacting with mostly idiosyncratic receptors. Among the entire set, a number of sequence-structure correlations can be identified, which may be used as initial hypotheses in predicting three-dimensional structures from primary sequences. Conversely, other sequence patterns are not predictive; for example, GAAA loop sequences and GG/CC receptors bind to each other with three distinct geometries. Finally, we observe an example of structural evolution in group II introns, in which loop-receptor motifs are substituted for each other while maintaining the larger three-dimensional geometry. Overall, the study gives a more complete view of RNA loop-helix interactions that exist in nature.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0049225PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494683PMC
May 2013

Biochemical and structural characterization of the GTP-preferring succinyl-CoA synthetase from Thermus aquaticus.

Acta Crystallogr D Biol Crystallogr 2012 Jul 15;68(Pt 7):751-62. Epub 2012 Jun 15.

Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.

Succinyl-CoA synthetase (SCS) from Thermus aquaticus was characterized biochemically via measurements of the activity of the enzyme and determination of its quaternary structure as well as its stability and refolding properties. The enzyme is most active between pH 8.0 and 8.4 and its activity increases with temperature to about 339 K. Gel-filtration chromatography and sedimentation equilibrium under native conditions demonstrated that the enzyme is a heterotetramer of two α-subunits and two β-subunits. The activity assays showed that the enzyme uses either ADP/ATP or GDP/GTP, but prefers GDP/GTP. This contrasts with Escherichia coli SCS, which uses GDP/GTP but prefers ADP/ATP. To understand the nucleotide preference, T. aquaticus SCS was crystallized in the presence of GDP, leading to the determination of the structure in complex with GDP-Mn(2+). A water molecule and Pro20β in T. aquaticus take the place of Gln20β in pig GTP-specific SCS, interacting well with the guanine base and other residues of the nucleotide-binding site. This leads to the preference for GDP/GTP, but does not hinder the binding of ADP/ATP.
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http://dx.doi.org/10.1107/S0907444912010852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3388811PMC
July 2012

X-linked sideroblastic anemia due to carboxyl-terminal ALAS2 mutations that cause loss of binding to the β-subunit of succinyl-CoA synthetase (SUCLA2).

J Biol Chem 2012 Aug 27;287(34):28943-55. Epub 2012 Jun 27.

Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, New York 10029, USA.

Mutations in the erythroid-specific aminolevulinic acid synthase gene (ALAS2) cause X-linked sideroblastic anemia (XLSA) by reducing mitochondrial enzymatic activity. Surprisingly, a patient with the classic XLSA phenotype had a novel exon 11 mutation encoding a recombinant enzyme (p.Met567Val) with normal activity, kinetics, and stability. Similarly, both an expressed adjacent XLSA mutation, p.Ser568Gly, and a mutation (p.Phe557Ter) lacking the 31 carboxyl-terminal residues also had normal or enhanced activity, kinetics, and stability. Because ALAS2 binds to the β subunit of succinyl-CoA synthetase (SUCLA2), the mutant proteins were tested for their ability to bind to this protein. Wild type ALAS2 bound strongly to a SUCLA2 affinity column, but the adjacent XLSA mutant enzymes and the truncated mutant did not bind. In contrast, vitamin B6-responsive XLSA mutations p.Arg452Cys and p.Arg452His, with normal in vitro enzyme activity and stability, did not interfere with binding to SUCLA2 but instead had loss of positive cooperativity for succinyl-CoA binding, an increased K(m) for succinyl-CoA, and reduced vitamin B6 affinity. Consistent with the association of SUCLA2 binding with in vivo ALAS2 activity, the p.Met567GlufsX2 mutant protein that causes X-linked protoporphyria bound strongly to SUCLA2, highlighting the probable role of an ALAS2-succinyl-CoA synthetase complex in the regulation of erythroid heme biosynthesis.
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http://dx.doi.org/10.1074/jbc.M111.306423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436539PMC
August 2012

Substitution for Asn460 cripples β-galactosidase (Escherichia coli) by increasing substrate affinity and decreasing transition state stability.

Arch Biochem Biophys 2012 May 22;521(1-2):51-61. Epub 2012 Mar 22.

Division of Biochemistry, Faculty of Science, University of Calgary, Calgary, AB, Canada T2N 1N4.

Substrate initially binds to β-galactosidase (Escherichia coli) at a 'shallow' site. It then moves ∼3Å to a 'deep' site and the transition state forms. Asn460 interacts in both sites, forming a water bridge interaction with the O3 hydroxyl of the galactosyl moiety in the shallow site and a direct H-bond with the O2 hydroxyl of the transition state in the deep site. Structural and kinetic studies were done with β-galactosidases with substitutions for Asn460. The substituted enzymes have enhanced substrate affinity in the shallow site indicating lower E·substrate complex energy levels. They have poor transition state stabilization in the deep site that is manifested by increased energy levels of the E·transition state complexes. These changes in stability result in increased activation energies and lower k(cat) values. Substrate affinity to N460D-β-galactosidase was enhanced through greater binding enthalpy (stronger H-bonds through the bridging water) while better affinity to N460T-β-galactosidase occurred because of greater binding entropy. The transition states are less stable with N460S- and N460T-β-galactosidase because of the weakening or loss of the important bond to the O2 hydroxyl of the transition state. For N460D-β-galactosidase, the transition state is less stable due to an increased entropy penalty.
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http://dx.doi.org/10.1016/j.abb.2012.03.014DOI Listing
May 2012

Ser-796 of β-galactosidase (Escherichia coli) plays a key role in maintaining a balance between the opened and closed conformations of the catalytically important active site loop.

Arch Biochem Biophys 2012 Jan 1;517(2):111-22. Epub 2011 Dec 1.

Division of Biochemistry, University of Calgary, Calgary, Alberta, Canada.

A loop (residues 794-803) at the active site of β-galactosidase (Escherichia coli) opens and closes during catalysis. The α and β carbons of Ser-796 form a hydrophobic connection to Phe-601 when the loop is closed while a connection via two H-bonds with the Ser hydroxyl occurs with the loop open. β-Galactosidases with substitutions for Ser-796 were investigated. Replacement by Ala strongly stabilizes the closed conformation because of greater hydrophobicity and loss of H-bonding ability while replacement with Thr stabilizes the open form through hydrophobic interactions with its methyl group. Upon substitution with Asp much of the defined loop structure is lost. The different open-closed equilibria cause differences in the stabilities of the enzyme·substrate and enzyme·transition state complexes and of the covalent intermediate that affect the activation thermodynamics. With Ala, large changes of both the galactosylation (k(2)) and degalactosylation (k(3)) rates occur. With Thr and Asp, the k(2) and k(3) were not changed as much but large ΔH(‡) and TΔS(‡) changes showed that the substitutions caused mechanistic changes. Overall, the hydrophobic and H-bonding properties of Ser-796 result in interactions strong enough to stabilize the open or closed conformations of the loop but weak enough to allow loop movement during the reaction.
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http://dx.doi.org/10.1016/j.abb.2011.11.017DOI Listing
January 2012

ADP-Mg2+ bound to the ATP-grasp domain of ATP-citrate lyase.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2011 Oct 24;67(Pt 10):1168-72. Epub 2011 Sep 24.

Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB T2N 1N4, Canada.

Human ATP-citrate lyase (EC 2.3.3.8) is the cytoplasmic enzyme that catalyzes the production of acetyl-CoA from citrate, CoA and ATP. The amino-terminal portion of the enzyme, containing residues 1-817, was crystallized in the presence of tartrate, ATP and magnesium ions. The crystals diffracted to 2.3 Å resolution. The structure shows ADP-Mg(2+) bound to the domain that possesses the ATP-grasp fold. The structure demonstrates that this crystal form could be used to investigate the structures of complexes with inhibitors of ATP-citrate lyase that bind at either the citrate- or ATP-binding site.
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http://dx.doi.org/10.1107/S1744309111028363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3212355PMC
October 2011

Catalytic role of the conformational change in succinyl-CoA:3-oxoacid CoA transferase on binding CoA.

Biochemistry 2010 Dec 11;49(48):10319-28. Epub 2010 Nov 11.

Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada.

Catalysis by succinyl-CoA:3-oxoacid CoA transferase proceeds through a thioester intermediate in which CoA is covalently linked to the enzyme. To determine the conformation of the thioester intermediate, crystals of the pig enzyme were grown in the presence of the substrate acetoacetyl-CoA. X-ray diffraction data show the enzyme in both the free form and covalently bound to CoA via Glu305. In the complex, the protein adopts a conformation in which residues 267-275, 280-287, 357-373, and 398-477 have shifted toward Glu305, closing the enzyme around the thioester. Enzymes provide catalysis by stabilizing the transition state relative to complexes with substrates or products. In this case, the conformational change allows the enzyme to interact with parts of CoA distant from the reactive thiol while the thiol is covalently linked to the enzyme. The enzyme forms stabilizing interactions with both the nucleotide and pantoic acid portions of CoA, while the interactions with the amide groups of the pantetheine portion are poor. The results shed light on how the enzyme uses the binding energy for groups remote from the active center of CoA to destabilize atoms closer to the active center, leading to acceleration of the reaction by the enzyme.
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http://dx.doi.org/10.1021/bi100659sDOI Listing
December 2010

Identification of the citrate-binding site of human ATP-citrate lyase using X-ray crystallography.

J Biol Chem 2010 Aug 17;285(35):27418-27428. Epub 2010 Jun 17.

Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada. Electronic address:

ATP-citrate lyase (ACLY) catalyzes the conversion of citrate and CoA into acetyl-CoA and oxaloacetate, coupled with the hydrolysis of ATP. In humans, ACLY is the cytoplasmic enzyme linking energy metabolism from carbohydrates to the production of fatty acids. In situ proteolysis of full-length human ACLY gave crystals of a truncated form, revealing the conformations of residues 2-425, 487-750, and 767-820 of the 1101-amino acid protein. Residues 2-425 form three domains homologous to the beta-subunit of succinyl-CoA synthetase (SCS), while residues 487-820 form two domains homologous to the alpha-subunit of SCS. The crystals were grown in the presence of tartrate or the substrate, citrate, and the structure revealed the citrate-binding site. A loop formed by residues 343-348 interacts via specific hydrogen bonds with the hydroxyl and carboxyl groups on the prochiral center of citrate. Arg-379 forms a salt bridge with the pro-R carboxylate of citrate. The pro-S carboxylate is free to react, providing insight into the stereospecificity of ACLY. Because this is the first structure of any member of the acyl-CoA synthetase (NDP-forming) superfamily in complex with its organic acid substrate, locating the citrate-binding site is significant for understanding the catalytic mechanism of each member, including the prototype SCS. Comparison of the CoA-binding site of SCSs with the similar structure in ACLY showed that ACLY possesses a different CoA-binding site. Comparisons of the nucleotide-binding site of SCSs with the similar structure in ACLY indicates that this is the ATP-binding site of ACLY.
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http://dx.doi.org/10.1074/jbc.M109.078667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2930740PMC
August 2010

Auxiliary Ca2+ binding sites can influence the structure of CIB1.

Protein Sci 2009 May;18(5):1128-34

Structural Biology Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.

Recent X-ray crystal structures and solution NMR spectroscopy data for calcium- and integrin-binding protein 1 (CIB1) have all revealed a common EF-hand domain structure for the protein. However, the orientation of the two protein domains, the oligomerization state, and the conformations of the N- and C-terminal extensions differ among the structures. In this study, we examine whether the binding of glutathione or auxiliary Ca(2+) ions as observed in the crystal structures, occur in solution, and whether these interactions can influence the structure or dimerization of CIB1. In addition, we test the potential phosphatase activity of CIB1, which was hypothesized based on the glutathione binding site geometry observed in one of the crystal structures of the protein. Biophysical and biochemical experiments failed to detect glutathione binding, protein dimerization, or phosphatase activity for CIB1 under several solution conditions. However, our data identify low affinity (K(d), 10(-2)M) Ca(2+) binding events that influence the structures of the N- and C-terminal extensions of CIB1 under high (300 mM) Ca(2+) crystallization conditions. In addition to providing a rationale for differences amongst the various solution and crystal structures of CIB1, our results show that the impact of low affinity Ca(2+) binding events should be considered when analyzing and interpreting protein crystallographic structures determined in the presence of very high Ca(2+) concentrations.
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http://dx.doi.org/10.1002/pro.104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2771315PMC
May 2009

Identification of the cysteine residue exposed by the conformational change in pig heart succinyl-CoA:3-ketoacid coenzyme A transferase on binding coenzyme A.

Biochemistry 2007 Sep 24;46(38):10852-63. Epub 2007 Aug 24.

Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) transfers CoA from succinyl-CoA to acetoacetate via a thioester intermediate with its active site glutamate residue, Glu 305. When CoA is linked to the enzyme, a cysteine residue can now be rapidly modified by 5,5'-dithiobis(2-nitrobenzoic acid), reflecting a conformational change of SCOT upon formation of the thioester. Since either Cys 28 or Cys 196 could be the target, each was mutated to Ser to distinguish between them. Like wild-type SCOT, the C196S mutant protein was modified rapidly in the presence of acyl-CoA substrates. In contrast, the C28S mutant protein was modified much more slowly under identical conditions, indicating that Cys 28 is the residue exposed on binding CoA. The specific activity of the C28S mutant protein was unexpectedly lower than that of wild-type SCOT. X-ray crystallography revealed that Ser adopts a different conformation than the native Cys. A chloride ion is bound to one of four active sites in the crystal structure of the C28S mutant protein, mimicking substrate, interacting with Lys 329, Asn 51, and Asn 52. On the basis of these results and the studies of the structurally similar CoA transferase from Escherichia coli, YdiF, bound to CoA, the conformational change in SCOT was deduced to be a domain rotation of 17 degrees coupled with movement of two loops: residues 321-329 that bury Cys 28 and interact with succinate or acetoacetate and residues 374-386 that interact with CoA. Modeling this conformational change has led to the proposal of a new mechanism for catalysis by SCOT.
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http://dx.doi.org/10.1021/bi700828hDOI Listing
September 2007

Participation of Cys123alpha of Escherichia coli succinyl-CoA synthetase in catalysis.

Acta Crystallogr D Biol Crystallogr 2007 Aug 17;63(Pt 8):876-84. Epub 2007 Jul 17.

Department of Biochemistry, University of Western Ontario, London, Ontario, Canada.

Succinyl-CoA synthetase has a highly conserved cysteine residue, Cys123alpha in the Escherichia coli enzyme, that is located near the CoA-binding site and the active-site histidine residue. To test whether the succinyl moiety of succinyl-CoA is transferred to the thiol of Cys123alpha as part of the catalytic mechanism, this residue was mutated to alanine, serine, threonine and valine. Each mutant protein was catalytically active, although less active than the wild type. This proved that the specific formation of a thioester bond with Cys123alpha is not part of the catalytic mechanism. To understand why the mutations affected catalysis, the crystal structures of the four mutant proteins were determined. The alanine mutant showed no structural changes yet had reduced activity, suggesting that the size of the cysteine is important for optimal activity. These results explain why this cysteine residue is conserved in the sequences of succinyl-CoA synthetases from different sources.
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http://dx.doi.org/10.1107/S0907444907029319DOI Listing
August 2007

Cloning, expression, purification, crystallization and preliminary X-ray analysis of Thermus aquaticus succinyl-CoA synthetase.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2007 May 14;63(Pt 5):399-402. Epub 2007 Apr 14.

Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.

Succinyl-CoA synthetase (SCS) is an enzyme of the citric acid cycle and is thus found in most species. To date, there are no structures available of SCS from a thermophilic organism. To investigate how the enzyme adapts to higher temperatures, SCS from Thermus aquaticus was cloned, overexpressed, purified and crystallized. Attempts to crystallize the enzyme were thwarted by proteolysis of the beta-subunit and preferential crystallization of the truncated form. Crystals of full-length SCS were grown after the purification protocol was modified to include frequent additions of protease inhibitors. The resulting crystals, which diffract to 2.35 A resolution, are of the protein in complex with Mn2+-GDP.
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http://dx.doi.org/10.1107/S1744309107017113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2335007PMC
May 2007

Beta-galactosidase (Escherichia coli) has a second catalytically important Mg2+ site.

Biochem Biophys Res Commun 2007 Jan 20;352(2):566-70. Epub 2006 Nov 20.

Biochemistry, Faculty of Science, University of Calgary, Calgary, Alta., Canada T2N 1N4.

It is shown here that Escherichia coli beta-galactosidase has a second Mg2+ binding site that is important for activity. Binding of Mg2+ to the second site caused the k(cat) (with oNPG as the substrate) to increase about 100 s(-1); the Km was not affected. The Kd for binding the second Mg2+ is about 10(-4)M. Since the concentration of free Mg2+ in E. coli is about 1-2 mM, the second site is physiologically significant. Non-polar substitutions (Ala or Leu) for Glu-797, a residue in an active site loop, eliminated the k(cat) increase. This indicates that the second Mg2+ site is near to Glu-797. The Ki values of transition state analogs were decreased by small but statistically significant amounts when the second Mg2+ site was occupied and Arrhenius plots showed that less entropic activation energy is required when the second site is occupied. These inhibitor and temperature results suggest that binding of the second Mg2+ helps to order the active site for stabilization of the transition state.
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http://dx.doi.org/10.1016/j.bbrc.2006.11.061DOI Listing
January 2007

Binding of adenine to Stx2, the protein toxin from Escherichia coli O157:H7.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2006 Jul 26;62(Pt 7):627-30. Epub 2006 Jun 26.

Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary AB T2N 1N4, Canada.

Stx2 is a protein toxin whose catalytic subunit acts as an N-glycosidase to depurinate a specific adenine base from 28S rRNA. In the holotoxin, the catalytic portion, A1, is linked to the rest of the A subunit, A2, and A2 interacts with the pentameric ring formed by the five B subunits. In order to test whether the holotoxin is active as an N-glycosidase, Stx2 was crystallized in the presence of adenosine and adenine. The crystals diffracted to approximately 1.8 angstroms and showed clear electron density for adenine in the active site. Adenosine had been cleaved, proving that Stx2 is an active N-glycosidase. While the holotoxin is active against small substrates, it would be expected that the B subunits would interfere with the binding of the 28S rRNA.
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http://dx.doi.org/10.1107/S1744309106021968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2242964PMC
July 2006

An assay for Fe(II)/2-oxoglutarate-dependent dioxygenases by enzyme-coupled detection of succinate formation.

Anal Biochem 2006 Jun 19;353(1):69-74. Epub 2006 Apr 19.

Department of Enzymology and Mechanistic Pharmacology, MMPD CEDD, GlaxoSmithKline, Collegeville, PA 19426, USA.

The Fe(II)/2-oxoglutarate-dependent dioxygenases are a catalytically diverse family of nonheme iron enzymes that oxidize their primary substrates while decomposing the 2-oxoglutarate cosubstrate to form succinate and CO(2). We report a generic assay for these enzymes that uses succinyl-coenzyme A synthetase, pyruvate kinase, and lactate dehydrogenase to couple the formation of the product succinate to the conversion of reduced nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide. We demonstrate the utility of this new method by measuring the kinetic parameters of two bacterial Fe(II)/2-oxoglutarate-dependent dioxygenases. Significantly, this method can be used to investigate both the productive turnover reactions and the nonproductive "uncoupled" decarboxylation reactions of this enzyme family, as demonstrated by using wild-type and variant forms of 2-oxoglutarate-dependent taurine dioxygenase. This assay is amenable to miniaturization and easily adapted to a format suitable for high-throughput screening; thus, it will be a valuable tool to study Fe(II)/2-oxoglutarate-dependent dioxygenases.
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http://dx.doi.org/10.1016/j.ab.2006.03.033DOI Listing
June 2006

Interactions of GTP with the ATP-grasp domain of GTP-specific succinyl-CoA synthetase.

J Biol Chem 2006 Apr 15;281(16):11058-65. Epub 2006 Feb 15.

Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.

Two isoforms of succinyl-CoA synthetase exist in mammals, one specific for ATP and the other for GTP. The GTP-specific form of pig succinyl-CoA synthetase has been crystallized in the presence of GTP and the structure determined to 2.1 A resolution. GTP is bound in the ATP-grasp domain, where interactions of the guanine base with a glutamine residue (Gln-20beta) and with backbone atoms provide the specificity. The gamma-phosphate interacts with the side chain of an arginine residue (Arg-54beta) and with backbone amide nitrogen atoms, leading to tight interactions between the gamma-phosphate and the protein. This contrasts with the structures of ATP bound to other members of the family of ATP-grasp proteins where the gamma-phosphate is exposed, free to react with the other substrate. To test if GDP would interact with GTP-specific succinyl-CoA synthetase in the same way that ADP interacts with other members of the family of ATP-grasp proteins, the structure of GDP bound to GTP-specific succinyl-CoA synthetase was also determined. A comparison of the conformations of GTP and GDP shows that the bases adopt the same position but that changes in conformation of the ribose moieties and the alpha- and beta-phosphates allow the gamma-phosphate to interact with the arginine residue and amide nitrogen atoms in GTP, while the beta-phosphate interacts with these residues in GDP. The complex of GTP with succinyl-CoA synthetase shows that the enzyme is able to protect GTP from hydrolysis when the active-site histidine residue is not in position to be phosphorylated.
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http://dx.doi.org/10.1074/jbc.M511785200DOI Listing
April 2006

Crystallographic trapping of the glutamyl-CoA thioester intermediate of family I CoA transferases.

J Biol Chem 2005 Dec 27;280(52):42919-28. Epub 2005 Oct 27.

Department of Biochemistry, McGill University, Quebec, Canada.

Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 A resolution, respectively. YdiF is organized into tetramers, with each monomer having an open alpha/beta structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent gamma-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases.
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http://dx.doi.org/10.1074/jbc.M510522200DOI Listing
December 2005

Cyclic nucleotide binding proteins in the Arabidopsis thaliana and Oryza sativa genomes.

BMC Bioinformatics 2005 Jan 11;6. Epub 2005 Jan 11.

Department of Biological Sciences, University of Calgary, AB, T2N 1N4 Canada.

Background: Cyclic nucleotides are ubiquitous intracellular messengers. Until recently, the roles of cyclic nucleotides in plant cells have proven difficult to uncover. With an understanding of the protein domains which can bind cyclic nucleotides (CNB and GAF domains) we scanned the completed genomes of the higher plants Arabidopsis thaliana (mustard weed) and Oryza sativa (rice) for the effectors of these signalling molecules.

Results: Our analysis found that several ion channels and a class of thioesterases constitute the possible cyclic nucleotide binding proteins in plants. Contrary to some reports, we found no biochemical or bioinformatic evidence for a plant cyclic nucleotide regulated protein kinase, suggesting that cyclic nucleotide functions in plants have evolved differently than in mammals.

Conclusion: This paper provides a molecular framework for the discussion of cyclic nucleotide function in plants, and resolves a longstanding debate about the presence of a cyclic nucleotide dependent kinase in plants.
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http://dx.doi.org/10.1186/1471-2105-6-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC545951PMC
January 2005

Structure of the CoA transferase from pig heart to 1.7 A resolution.

Acta Crystallogr D Biol Crystallogr 2004 Oct 23;60(Pt 10):1717-25. Epub 2004 Sep 23.

Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT; EC 2.8.3.5) activates the acetoacetate in ketone bodies by transferring the CoA group from succinyl-CoA to acetoacetate to produce acetoacetyl-CoA and succinate. In the reaction, a glutamate residue at the active site of the enzyme forms a thioester bond with CoA and in this form the enzyme is subject to autolytic fragmentation. The crystal structure of pig heart SCOT has been solved and refined to 1.7 A resolution in a new crystal form. The structure shows the active-site glutamate residue in a conformation poised for autolytic fragmentation, with its side chain accepting one hydrogen bond from Asn281 and another from its own amide N atom. However, the conformation of this glutamate side chain would have to change for the residues that are conserved in the CoA transferases (Gln99, Gly386 and Ala387) to participate in stabilizing the tetrahedral transition states of the catalytic mechanism. The structures of a deletion mutant in two different crystal forms were also solved.
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http://dx.doi.org/10.1107/S0907444904017974DOI Listing
October 2004

Structure of shiga toxin type 2 (Stx2) from Escherichia coli O157:H7.

J Biol Chem 2004 Jun 9;279(26):27511-7. Epub 2004 Apr 9.

Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.

Several serotypes of Escherichia coli produce protein toxins closely related to Shiga toxin (Stx) from Shigella dysenteriae serotype 1. These Stx-producing E. coli cause outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in humans, with the latter being more likely if the E. coli produce Stx2 than if they only produce Stx1. To investigate the differences among the Stxs, which are all AB(5) toxins, the crystal structure of Stx2 from E. coli O157:H7 was determined at 1.8-A resolution and compared with the known structure of Stx. Our major finding was that, in contrast to Stx, the active site of the A-subunit of Stx2 is accessible in the holotoxin, and a molecule of formic acid and a water molecule mimic the binding of the adenine base of the substrate. Further, the A-subunit adopts a different orientation with respect to the B-subunits in Stx2 than in Stx, due to interactions between the carboxyl termini of the B-subunits and neighboring regions of the A-subunit. Of the three types of receptor-binding sites in the B-pentamer, one has a different conformation in Stx2 than in Stx, and the carboxyl terminus of the A-subunit binds at another. Any of these structural differences might result in different mechanisms of action of the two toxins and the development of hemolytic uremic syndrome upon exposure to Stx2.
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http://dx.doi.org/10.1074/jbc.M401939200DOI Listing
June 2004

Structure of the mammalian CoA transferase from pig heart.

Biochemistry 2002 Dec;41(49):14455-62

Department of Biochemistry, University of Western Ontario, London, Ontario, Canada, N6A 5C1.

Ketoacidosis affects patients who are deficient in the enzyme activity of succinyl-CoA:3-ketoacid CoA transferase (SCOT), since SCOT catalyses the activation of acetoacetate in the metabolism of ketone bodies. Thus far, structure/function analysis of the mammalian enzyme has been predicted based on the three-dimensional structure of a CoA transferase determined from an anaerobic bacterium that utilizes its enzyme for glutamate fermentation. To better interpret clinical data, we have determined the structure of a mammalian CoA transferase from pig heart by X-ray crystallography to 2.5 A resolution. Instrumental to the structure determination were selenomethionine substitution and the use of argon during purification and crystallization. Although pig heart SCOT adopts an alpha/beta protein fold, resembling the overall fold of the bacterial CoA transferase, several loops near the active site of pig heart SCOT follow different paths than the corresponding loops in the bacterial enzyme, accounting for differences in substrate specificities. Two missense mutations found associated with SCOT of ketoacidosis patients were mapped to a location in the structure that might disrupt the stabilization of the amino-terminal strand and thereby interfere with the proper folding of the protein into a functional enzyme.
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http://dx.doi.org/10.1021/bi020568fDOI Listing
December 2002

Two glutamate residues, Glu 208 alpha and Glu 197 beta, are crucial for phosphorylation and dephosphorylation of the active-site histidine residue in succinyl-CoA synthetase.

Biochemistry 2002 Jan;41(2):537-46

Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.

Succinyl-CoA synthetase catalyzes the reversible reaction succinyl-CoA + NDP + P(i) <--> succinate + CoA + NTP (N denoting adenosine or guanosine). The enzyme consists of two different subunits, designated alpha and beta. During the reaction, a histidine residue of the alpha-subunit is transiently phosphorylated. This histidine residue interacts with Glu 208 alpha at site I in the structures of phosphorylated and dephosphorylated Escherichia coli SCS. We postulated that Glu 197 beta, a residue in the nucleotide-binding domain, would provide similar stabilization of the histidine residue during the actual phosphorylation/dephosphorylation by nucleotide at site II. In this work, these two glutamate residues have been mutated individually to aspartate or glutamine. Glu 197 beta has been additionally mutated to alanine. The mutant proteins were tested for their ability to be phosphorylated in the forward or reverse direction. The aspartate mutant proteins can be phosphorylated in either direction, while the E208 alpha Q mutant protein can only be phosphorylated by NTP, and the E197 beta Q mutant protein can only be phosphorylated by succinyl-CoA and P(i). These results demonstrate that the length of the side chain at these positions is not critical, but that the charge is. Most significantly, the E197 beta A mutant protein could not be phosphorylated in either direction. Its crystal structure shows large differences from the wild-type enzyme in the conformation of two residues of the alpha-subunit, Cys 123 alpha-Pro 124 alpha. We postulate that in this conformation, the protein cannot productively bind succinyl-CoA for phosphorylation via succinyl-CoA and P(i).
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http://dx.doi.org/10.1021/bi011518yDOI Listing
January 2002