Pig GTP-specific succinyl-CoA synthetase in complex with succinate

2014 ◽  
Vol 70 (a1) ◽  
pp. C1650-C1650
Author(s):  
Ji Huang ◽  
Marie Fraser
Keyword(s):  
Binding Site ◽  
Catalytic Mechanism ◽  
Methylmalonic Aciduria ◽  
Β Subunit ◽  
Α Subunit ◽  
Conserved Sequence ◽  
C Terminus ◽  
Mitochondrial Dna Depletion ◽  
Nucleotide Specificity ◽  
Succinyl Coa Synthetase

Succinyl-CoA synthetase (SCS) exists in the mitochondria of mammals as two different isoforms; one is ATP-specific and the other is GTP-specific. SCS is a heterodimer, and the two isoforms have a common α-subunit, but different β-subunits [1]. The β-subunit determines nucleotide specificity. Mutations in the α-subunit or the ATP-specific β-subunit can cause encephalomyopathy due to mitochondrial DNA depletion, along with lactic acidosis and methylmalonic aciduria (reviewed in [2]). The reaction catalyzed by SCS, succinyl-CoA+ NDP + Pi⇌succinate +CoA + NTP, is reversible, and the direction depends on the relative concentrations of substrates and products. Only after all substrate-binding sites are discovered can the catalytic mechanism of SCS be fully understood. Structures of SCS with ADP, GDP, GTP, Pi and CoA have been determined, but the succinate-binding site, or the binding site for the succinyl-portion of succinyl-CoA, is still unknown. Succinate is predicted to bind to the conserved sequence Gly-Gly-Ile-Val (327β-330β) located in a loop of the β-subunit of GTP-specific SCS. Crystals of other complexes with pig GTP-specific SCS have diffracted well, so we are crystallizing this enzyme in complex with succinate. Initially, plasmid containing the genes encoding pig GTP-specific SCS was transformed into E coli. After overproducing the desired protein with a 6-His tag on the C-terminus of the α-subunit, three different purification columns were used to obtain the GTP-SCS protein at high purity. Succinate was then co-crystallized with GTP-SCS under conditions containing polyethylene glycol 3350, magnesium formate and HEPES, pH 7.0.

scholarly journals Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants

2008 ◽  
Vol 40 (4) ◽  
pp. 185-198 ◽  
Author(s):  
Sébastien Legardinier ◽  
Jean-Claude Poirier ◽  
Danièle Klett ◽  
Yves Combarnous ◽  
Claire Cahoreau
Keyword(s):  
Thermal Stability ◽  
Chorionic Gonadotropin ◽  
Sf9 Cells ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
C Terminus ◽  
N Terminus ◽  
Covalently Linked

Recombinant equine LH/chorionic gonadotropin (eLH/CG) was expressed in the baculovirus–Sf9 insect cell system either as a single-chain with the C-terminus of the β-subunit fused to the N-terminus of the α-subunit or as non-covalently linked heterodimers with or without a polyhistidine tag at various locations. All these non-covalently linked eLH/CG variants were secreted as stable heterodimers in the medium of infected Sf9 cells. To assess the influence of the presence and the position of polyhistidine tag on LH bioactivity, we expressed four non-covalently linked tagged heterodimeric eLH/CG variants that were secreted in threefold higher quantities than the single chain. Among them, only two exhibited full in vitro LH bioactivity, relative to untagged heterodimers, namely the one His-tagged at the N-terminus of α-subunit and the other at the C-terminus of the β-subunit both of which are amenable to nickel-affinity purification. Furthermore, single-chain eLH/CG was found to be N- and O-glycosylated but nevertheless less active in in vitro LH bioassays than natural eCG and heterodimeric recombinant eLH/CG. The thermal stability of natural and recombinant hormones was assessed by the initial rates of dissociation from 20 to 90 °C. Heterodimeric eLH/CG from Sf9 cells was found to be as stable as pituitary eLH and serum eCG (T1/2, 74–77 °C). Although Sf9 cells only elaborated short immature-type carbohydrate side chains on glycoproteins, recombinant eLH/CG produced in these cells exhibited stabilities similar to that of pituitary eLH. In conclusion, recombinant heterodimeric eLH/CG exhibits the same thermal stability as natural pituitary LH and its advantages over the single-chain eLH/CG include higher secretion, higher in vitro bioactivity, and reduced potential risk of immunogenicity.

scholarly journals Two transgenic mouse models for β-subunit components of succinate-CoA ligase yielding pleiotropic metabolic alterations

2016 ◽  
Vol 473 (20) ◽  
pp. 3463-3485 ◽  
Author(s):  
Gergely Kacso ◽  
Dora Ravasz ◽  
Judit Doczi ◽  
Beáta Németh ◽  
Ory Madgar ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Metabolic Pathways ◽  
Β Subunit ◽  
Transgenic Mouse Models ◽  
Wild Type ◽  
Α Subunit ◽  
Coa Ligase ◽  
Mitochondrial Dna Depletion ◽  
Substrate Level ◽  
Age Dependent

Succinate-CoA ligase (SUCL) is a heterodimer enzyme composed of Suclg1 α-subunit and a substrate-specific Sucla2 or Suclg2 β-subunit yielding ATP or GTP, respectively. In humans, the deficiency of this enzyme leads to encephalomyopathy with or without methylmalonyl aciduria, in addition to resulting in mitochondrial DNA depletion. We generated mice lacking either one Sucla2 or Suclg2 allele. Sucla2 heterozygote mice exhibited tissue- and age-dependent decreases in Sucla2 expression associated with decreases in ATP-forming activity, but rebound increases in cardiac Suclg2 expression and GTP-forming activity. Bioenergetic parameters including substrate-level phosphorylation (SLP) were not different between wild-type and Sucla2 heterozygote mice unless a submaximal pharmacological inhibition of SUCL was concomitantly present. mtDNA contents were moderately decreased, but blood carnitine esters were significantly elevated. Suclg2 heterozygote mice exhibited decreases in Suclg2 expression but no rebound increases in Sucla2 expression or changes in bioenergetic parameters. Surprisingly, deletion of one Suclg2 allele in Sucla2 heterozygote mice still led to a rebound but protracted increase in Suclg2 expression, yielding double heterozygote mice with no alterations in GTP-forming activity or SLP, but more pronounced changes in mtDNA content and blood carnitine esters, and an increase in succinate dehydrogenase activity. We conclude that a partial reduction in Sucla2 elicits rebound increases in Suclg2 expression, which is sufficiently dominant to overcome even a concomitant deletion of one Suclg2 allele, pleiotropically affecting metabolic pathways associated with SUCL. These results as well as the availability of the transgenic mouse colonies will be of value in understanding SUCL deficiency.

ATP-specificity of succinyl-CoA synthetase fromBlastocystis hominis

2019 ◽  
Vol 75 (7) ◽  
pp. 647-659
Author(s):  
Ji Huang ◽  
Vinh H. Nguyen ◽  
Karleigh A. Hamblin ◽  
Robin Maytum ◽  
Mark van der Giezen ◽  
...  
Keyword(s):  
Binding Site ◽  
Electrostatic Interactions ◽  
Structural Changes ◽  
Tricarboxylic Acid Cycle ◽  
Side Chain ◽  
Blastocystis Hominis ◽  
Mutant Proteins ◽  
Nucleotide Specificity ◽  
Better Than ◽  
Succinyl Coa Synthetase

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 Mg2+-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 ofBlastocystis hominisSCS binds GTP better than ATP according to kinetic assays, only the complex with Mg2+-ADP was obtained.

scholarly journals Spectrin αII and βII isoforms interact with high affinity at the tetramerization site

2003 ◽  
Vol 374 (3) ◽  
pp. 613-624 ◽  
Author(s):  
Paola A. BIGNONE ◽  
Anthony J. BAINES
Keyword(s):  
Splice Variants ◽  
Entropy Change ◽  
Cell Junctions ◽  
Β Subunit ◽  
Α Subunit ◽  
High Affinity ◽  
Enthalpy Changes ◽  
C Terminus ◽  
Kd Values ◽  
Erythroid Spectrin

Spectrin tetramers form by the interaction of two α–β dimers through two helices close to the C-terminus of a β subunit and a single helix at the N-terminus of an α subunit. Early work on spectrin from solid tissues (typified by αII and βII polypeptides) indicated that it forms a more stable tetramer than erythroid spectrin (αI–βI). In the present study, we have probed the molecular basis of this phenomenon. We have quantified the interactions of N-terminal regions of two human α polypeptides (αI and αII) with the C-terminal regions of three β isoforms (βIΣ1, βIIΣ1 and βIIΣ2). αII binds either βII form with a much higher affinity than αI binds βIΣ1 (Kd values of 5–9 nM and 840 nM respectively at 25 °C). βIIΣ1 and βIIΣ2 are splice variants with different C-terminal extensions outside the tetramerization site: these extensions affect the rate rather than the affinity of α subunit interaction. αII spectrin interacts with each β subunit with higher affinity than αI, and the βII polypeptides have higher affinities for both α chains than βIΣ1. The first full repeat of the α subunit has a major role in determining affinity. Enthalpy changes in the αII–βIIΣ2 interaction are large, but the entropy change is comparatively small. The interaction is substantially reduced, but not eliminated, by concentrated salt solutions. The high affinity and slow overall kinetics of association and dissociation of αII–βII spectrin may suit it well to a role in strengthening cell junctions and providing stable anchor points for transmembrane proteins at points specified by cell-adhesion molecules.

scholarly journals The Small Subunit of M · AquI Is Responsible for Sequence-Specific DNA Recognition and Binding in the Absence of the Catalytic Domain

2003 ◽  
Vol 185 (4) ◽  
pp. 1284-1288 ◽  
Author(s):  
Hatice Pinarbasi ◽  
Ergun Pinarbasi ◽  
David P. Hornby
Keyword(s):  
Dna Binding ◽  
Dna Sequence ◽  
Dna Methyltransferase ◽  
Dna Recognition ◽  
Dna Methyltransferases ◽  
Binary Complex ◽  
Β Subunit ◽  
Α Subunit ◽  
Conserved Sequence ◽  
Covalent Complex

ABSTRACT AquI DNA methyltransferase (M · AquI) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5′-CCCGGG-3′. M · AquI is a heterodimer in which the polypeptide chain is separated at the junction between the two equivalent structural domains in the related enzyme M · HhaI. Recently, we reported the subcloning, overexpression, and purification of the subunits (α and β) of M · AquI separately. Here we describe the DNA binding properties of M · AquI. The results presented here indicate that the β subunit alone contains all of the information for sequence-specific DNA recognition and binding. The first step in the sequence-specific recognition of DNA by M · AquI involves the formation of binary complex with the target recognition domain in conjunction with conserved sequence motifs IX and X, found in all known C5 DNA methyltransferases, contained in the β subunit. The α subunit enhances the binding of the β subunit to DNA specifically and nonspecifically. It is likely that the addition of the α subunit to the β subunit stabilizes the conformation of the β subunit and thereby enhances its affinity for DNA indirectly. Addition of S-adenosyl-l-methionine and its analogues S-adenosyl-l-homocysteine and sinefungin enhances binding, but only in the presence of the α subunit. These compounds did not have any effect on DNA binding by the β subunit alone. Using a 30-mer oligodeoxynucleotide substrate containing 5-fluorodeoxycytidine (5-FdC), it was found that the β subunit alone did not form a covalent complex with its specific sequence in the absence or presence of S-adenosyl-l-methionine. However, the addition of the α subunit to the β subunit led to the formation of a covalent complex with specific DNA sequence containing 5-FdC.

scholarly journals Truncated Human ATP-Specific Succinyl-CoA Synthetase

2014 ◽  
Vol 70 (a1) ◽  
pp. C1641-C1641
Author(s):  
Marie Fraser ◽  
Chelsea Yu ◽  
Koto Hayakawa ◽  
Koto Hayakawa
Keyword(s):  
Aminolevulinic Acid ◽  
Ketone Bodies ◽  
Stop Codon ◽  
Reverse Direction ◽  
Β Subunit ◽  
Sequence Comparisons ◽  
Α Subunit ◽  
Heme Synthesis ◽  
Carboxy Terminal ◽  
Succinyl Coa Synthetase

Two isoforms of the heterodimeric enzyme succinyl-CoA synthetase (SCS) exist in the mitochondria of humans. One is specific for ATP, while the other is specific for GTP. Both catalyze the reversible reaction: succinate + CoA + NTP ⇌ succinyl-CoA + NDP + Pi, where N denotes adenosine or guanosine. SCS is best known as an enzyme of the citric acid cycle where the reaction generates NTP. In the reverse direction, SCS replenishes succinyl-CoA required for the catabolism of ketone bodies and for heme synthesis. Nucleotide-specific forms are thought to be required for SCS to serve its different metabolic roles. The nucleotide specificity lies in the β-subunit [1], and the β-subunit of human ATP-specific SCS has been shown to interact with the C-terminus of erythroid-specific aminolevulinic acid synthase (ALAS2) [2]. ALAS2 catalyzes the committed step in heme synthesis: succinyl-CoA + Gly ⇌ 5-aminolevulinate + CoA + CO2. An interaction between SCS and ALAS2 makes biological sense, since this could provide channeling of succinyl-CoA from SCS to ALAS2. We hypothesize that the interaction is with the carboxy-terminus of the β-subunit of ATP-specific SCS because sequence comparisons show that the β-subunit of ATP-specific SCS has a carboxy-terminal extension when compared to other SCSs' β-subunits. To test this hypothesis, we added a carboxy-terminal His8-tag to the α-subunit of human ATP-specific SCS and mutated the codon for Thr 396β to a stop codon. This truncated version of human ATP-specific SCS has been produced in E. coli and purified. As well as testing to see if truncated human ATP-specific SCS interacts with ALAS2, we are using the truncated version in crystallization trials. Crystals of full-length human ATP-specific SCS diffract to only 3.2 Å and our goal is to obtain better-diffracting crystals of the complex of ATP with truncated human ATP-specific SCS.

scholarly journals Crystal Structure of Adenylylsulfate Reductase from Desulfovibrio gigas Suggests a Potential Self-Regulation Mechanism Involving the C Terminus of the β-Subunit

2009 ◽  
Vol 191 (24) ◽  
pp. 7597-7608 ◽  
Author(s):  
Yuan-Lan Chiang ◽  
Yin-Cheng Hsieh ◽  
Jou-Yin Fang ◽  
En-Hong Liu ◽  
Yen-Chieh Huang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Active Site ◽  
Substrate Binding ◽  
Regulation Mechanism ◽  
Β Subunit ◽  
Α Subunit ◽  
Desulfovibrio Gigas ◽  
C Terminus ◽  
Terminal Loop

ABSTRACT Adenylylsulfate reductase (adenosine 5′-phosphosulfate [APS] reductase [APSR]) plays a key role in catalyzing APS to sulfite in dissimilatory sulfate reduction. Here, we report the crystal structure of APSR from Desulfovibrio gigas at 3.1-Å resolution. Different from the α2β2-heterotetramer of the Archaeoglobus fulgidus, the overall structure of APSR from D. gigas comprises six αβ-heterodimers that form a hexameric structure. The flavin adenine dinucleotide is noncovalently attached to the α-subunit, and two [4Fe-4S] clusters are enveloped by cluster-binding motifs. The substrate-binding channel in D. gigas is wider than that in A. fulgidus because of shifts in the loop (amino acid 326 to 332) and the α-helix (amino acid 289 to 299) in the α-subunit. The positively charged residue Arg160 in the structure of D. gigas likely replaces the role of Arg83 in that of A. fulgidus for the recognition of substrates. The C-terminal segment of the β-subunit wraps around the α-subunit to form a functional unit, with the C-terminal loop inserted into the active-site channel of the α-subunit from another αβ-heterodimer. Electrostatic interactions between the substrate-binding residue Arg282 in the α-subunit and Asp159 in the C terminus of the β-subunit affect the binding of the substrate. Alignment of APSR sequences from D. gigas and A. fulgidus shows the largest differences toward the C termini of the β-subunits, and structural comparison reveals notable differences at the C termini, activity sites, and other regions. The disulfide comprising Cys156 to Cys162 stabilizes the C-terminal loop of the β-subunit and is crucial for oligomerization. Dynamic light scattering and ultracentrifugation measurements reveal multiple forms of APSR upon the addition of AMP, indicating that AMP binding dissociates the inactive hexamer into functional dimers, presumably by switching the C terminus of the β-subunit away from the active site. The crystal structure of APSR, together with its oligomerization properties, suggests that APSR from sulfate-reducing bacteria might self-regulate its activity through the C terminus of the β-subunit.

The Human Follitropin α-Subunit C Terminus Collaborates with a β-Subunit Cystine Noose and an α-Subunit Loop To Assemble a Receptor-Binding Domain Competent for Signal Transduction†

Biochemistry ◽  
1998 ◽  
Vol 37 (7) ◽  
pp. 1762-1768 ◽  
Author(s):  
Carrie J. Arnold ◽  
Cheng Liu ◽  
Barbara Lindau-Shepard ◽  
Michele L. Losavio ◽  
Maria T. Patrascu ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Receptor Binding ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Β Subunit ◽  
Α Subunit ◽  
Subunit C ◽  
C Terminus

scholarly journals Discovery of fungal surface NADases predominantly present in pathogenic species

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Øyvind Strømland ◽  
Juha P. Kallio ◽  
Annica Pschibul ◽  
Renate H. Skoge ◽  
Hulda M. Harðardóttir ◽  
...  
Keyword(s):  
Cell Death ◽  
Aspergillus Fumigatus ◽  
Neurospora Crassa ◽  
Binding Site ◽  
Host Cell ◽  
Nicotinamide Adenine Dinucleotide ◽  
Catalytic Mechanism ◽  
Pathogenic Species ◽  
X Ray ◽  
Cellular Bioenergetics

AbstractNicotinamide adenine dinucleotide (NAD) is a key molecule in cellular bioenergetics and signalling. Various bacterial pathogens release NADase enzymes into the host cell that deplete the host’s NAD+ pool, thereby causing rapid cell death. Here, we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa. The enzymes harbour a tuberculosis necrotizing toxin (TNT) domain and are predominately present in pathogenic species. The 1.6 Å X-ray structure of the homodimeric A. fumigatus protein reveals unique properties including N-linked glycosylation and a Ca2+-binding site whose occupancy regulates activity. The structure in complex with a substrate analogue suggests a catalytic mechanism that is distinct from those of known NADases, ADP-ribosyl cyclases and transferases. We propose that fungal NADases may convey advantages during interaction with the host or competing microorganisms.

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