Papers by Cheryl Ingram-Smith
Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key p... more Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PPi-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant E. histolytica ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred sub-strate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred sub-strate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PPi were found to inhibit the acetate-forming direction of the reaction, with 50 % inhibito...
doi:10.1155/2012/509579 Research Article Role of Motif III in Catalysis by Acetyl-CoA Synthetase
Copyright © 2012 Cheryl Ingram-Smith et al. This is an open access article distributed under the ... more Copyright © 2012 Cheryl Ingram-Smith et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The acyl-adenylate-forming enzyme superfamily, consisting of acyl- and aryl-CoA synthetases, the adenylation domain of the nonribosomal peptide synthetases, and luciferase, has three signature motifs (I–III) and ten conserved core motifs (A1–A10), some of which overlap the signature motifs. The consensus sequence for signature motif III (core motif A7) in acetyl-CoA synthetase is Y-X-S/T/A-G-D, with an invariant fifth position, highly conserved first and fourth positions, and variable second and third positions. Kinetic studies of enzyme variants revealed that an alteration at any position resulted in a strong decrease in the catalytic rate, although the most deleterious effects were observed when the first or fifth positions we...
This article cites 39 articles, 17 of which can be accessed free
Phosphotransacetylase Class IIa Enzyme from Phytophthora ramorum
Biochemistry and Genetics
High-Yield Facts Storage and Expression of Genetic Information DNA Structure, Replication, and Re... more High-Yield Facts Storage and Expression of Genetic Information DNA Structure, Replication, and Repair Gene Expression Gene Regulation Acid-Base Equilibria, Amino Acids, and Protein Structure/FunctionAcid-Base Equilibria, Amino Acids, and Protein StructureProtein Structure/Function Intermediary Metabolism Carbohydrate Metabolism Bioenergetics and Energy Metabolism Amino Acid, Lipid, and Nucleotide Metabolism Nutrition Vitamins and Minerals Hormones and Integrated Metabolism Inheritance Mechanisms and Biochemical Genetics Inheritance Mechanisms/Risk Calculations Genetic and Biochemical Diagnosis Appendix: Summary of Questions as Related to the USMLE Content Outline Bibliography Index
Journal of Bacteriology, 1998
Acetate kinase catalyzes the reversible phosphorylation of acetate (CH 3 COO ؊ ؉ ATP^CH 3 CO 2 PO... more Acetate kinase catalyzes the reversible phosphorylation of acetate (CH 3 COO ؊ ؉ ATP^CH 3 CO 2 PO 3 2؊ ؉ ADP). A mechanism which involves a covalent phosphoryl-enzyme intermediate has been proposed, and chemical modification studies of the enzyme from Escherichia coli indicate an unspecified glutamate residue is phosphorylated (J. A. Todhunter and D. L. Purich, Biochem. Biophys. Res. Commun. 60:273-280, 1974). Alignment of the amino acid sequences for the acetate kinases from E. coli (Bacteria domain), Methanosarcina thermophila (Archaea domain), and four other phylogenetically divergent microbes revealed high identity which included five glutamates. These glutamates were replaced in the M. thermophila enzyme to determine if any are essential for catalysis. The histidine-tagged altered enzymes were produced in E. coli and purified to electrophoretic homogeneity by metal affinity chromatography. Replacements of E384 resulted in either undetectable or extremely low kinase activity, suggesting E384 is essential for catalysis which supports the proposed mechanism. Replacement of E385 influenced the K m values for acetate and ATP with only moderate decreases in k cat , which suggests that this residue is involved in substrate binding but not catalysis. The unaltered acetate kinase was not inactivated by N-ethylmaleimide; however, replacement of E385 with cysteine conferred sensitivity to N-ethylmaleimide which was prevented by preincubation with acetate, acetyl phosphate, ATP, or ADP, suggesting that E385 is located near the active site. Replacement of E97 decreased the K m value for acetate but not ATP, suggesting this residue is involved in binding acetate. Replacement of either E32 or E334 had no significant effects on the kinetic constants, which indicates that neither residue is essential for catalysis or significantly influences the binding of acetate or ATP.
2.1 A structure of acyl-adenylate synthetase from Methanosarcina acetivorans containing a link between Lys256 and Cys298
FEBS letters, Jan 27, 2017
ADP-forming acetyl-CoA synthetase (ACD) catalyzes the interconversion of acetyl-CoA and acetate. ... more ADP-forming acetyl-CoA synthetase (ACD) catalyzes the interconversion of acetyl-CoA and acetate. The related succinyl-CoA synthetase follows a three-step mechanism involving a single phosphoenzyme, but a novel four-step mechanism with two phosphoenzyme intermediates was proposed for Pyrococcus ACD. Characterization of enzyme variants of Entamoeba ACD in which the two proposed phosphorylated His residues were individually altered revealed that only His252 is essential for enzymatic activity. Analysis of variants altered at two residues proposed to interact with the phosphohistidine loop that swings between distinct parts of the active site are consistent with a mechanism involving a single phosphoenzyme intermediate. Our results suggest ACDs with different subunit structures may employ slightly different mechanisms to bridge the span between active sites I and II.
Eukaryotic Cell, 2015
Phosphotransacetylase (Pta), a key enzyme in bacterial metabolism, catalyzes the reversible trans... more Phosphotransacetylase (Pta), a key enzyme in bacterial metabolism, catalyzes the reversible transfer of an acetyl group from acetyl phosphate to coenzyme A (CoA) to produce acetyl-CoA and Pi. Two classes of Pta have been identified based on the absence (PtaI) or presence (PtaII) of an N-terminal regulatory domain. PtaIhas been fairly well studied in bacteria and one genus of archaea; however, only theEscherichia coliandSalmonella entericaPtaIIenzymes have been biochemically characterized, and they are allosterically regulated. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Pta from the oomycetePhytophthora ramorum. The two Ptas fromP. ramorum, designated PrPtaII1 and PrPtaII2, both belong to class II. PrPtaII1 displayed positive cooperativity for both acetyl phosphate and CoA and is allosterically regulated. We compared the effects of different metabolites on PrPtaII1 and theS. entericaPtaIIand found that, although the N-terminal regulatory doma...
Life (Basel, Switzerland), Jan 12, 2015
Acetate kinase (ACK), which catalyzes the reversible phosphorylation of acetate by ATP, is a memb... more Acetate kinase (ACK), which catalyzes the reversible phosphorylation of acetate by ATP, is a member of the acetate and sugar kinase/heat shock cognate/actin (ASKHA) superfamily. ASKHA family members share a common core fold that includes an ATPase domain with five structural motifs. The PHOSPHATE1 motif has previously been shown to be important for catalysis. We have investigated the role of two of these motifs in the Methanosarcina thermophila ACK (MtACK) and have shown that residues projecting into the ACK active site from the PHOSPHATE2 and ADENOSINE loops and a third highly conserved loop designated here as LOOP3 play key roles in nucleotide triphosphate (NTP) selection and utilization. Alteration of Asn211 of PHOSPHATE2, Gly239 of LOOP3, and Gly331 of ADENOSINE greatly reduced catalysis. In particular, Gly331, which is highly conserved throughout the ASKHA superfamily, has the greatest effect on substrate selection. Alteration at this site strongly skewed MtACK toward utilizati...
The Plant cell, 2014
Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.... more Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substa...
Trends in Microbiology, 2007
Protein Science, 2008
The unique biochemical properties of acetate kinase present a classic conundrum in the study of t... more The unique biochemical properties of acetate kinase present a classic conundrum in the study of the mechanism of enzyme-catalyzed phosphoryl transfer. Large, single crystals of acetate kinase from Methanosarcina thennophila were grown from a solution of ammonium sulfate in the presence of ATP. The crystals diffract to beyond 1.7 8, resolution. Analysis of X-ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 A, b = 67 8,, c = 83 A, p = 103". Diffraction data have been collected from the crystals at 110 and 277 K. Data collected at 277 K extend to lower resolution, but are more reproducible. The orientation of a noncrystallographic twofold axis of symmetry has been determined. Based on an analysis of the predicted amino acid sequences of acetate kinase from several organisms, we hypothesize that acetate kinase is a member of the sugar kinase/actin/hsp70 structural family.
Journal of Structural Biology, 2013
Acetate kinases (ACKs) are members of the acetate and sugar kinase/hsp70/actin (ASKHA) superfamil... more Acetate kinases (ACKs) are members of the acetate and sugar kinase/hsp70/actin (ASKHA) superfamily and catalyze the reversible phosphorylation of acetate, with ADP/ATP the most common phosphoryl acceptor/donor. While prokaryotic ACKs have been the subject of extensive biochemical and structural characterization, there is a comparative paucity of information on eukaryotic ACKs, and prior to this report, no structure of an ACK of eukaryotic origin was available. We determined the structures of ACKs from the eukaryotic pathogens Entamoeba histolytica and Cryptococcus neoformans. Each active site is located at an interdomain interface, and the acetate and phosphate binding pockets display sequence and structural conservation with their prokaryotic counterparts. Interestingly, the E. histolytica ACK has previously been shown to be pyrophosphate (PP i)-dependent, and is the first ACK demonstrated to have this property. Examination of its structure demonstrates how subtle amino acid substitutions within the active site have converted cosubstrate specificity from ATP to PP i while retaining a similar backbone conformation. Differences in the angle between domains surrounding the active site suggest that interdomain movement may accompany catalysis. Taken together, these structures are consistent with the eukaryotic ACKs following a similar reaction mechanism as is proposed for the prokaryotic homologs.
Journal of Biological Chemistry, 2000
The role of histidine in the catalytic mechanism of acetate kinase from Methanosarcina thermophil... more The role of histidine in the catalytic mechanism of acetate kinase from Methanosarcina thermophila was investigated by diethylpyrocarbonate inactivation and site-directed mutagenesis. Inactivation was accompanied by an increase in absorbance at 240 nm with no change in absorbance at 280 nm, and treatment of the inactivated enzyme with hydroxylamine restored 95% activity, results that indicated diethylpyrocarbonate inactivates the enzyme by the specific modification of histidine. The substrates ATP, ADP, acetate, and acetyl phosphate protected against inactivation suggesting at least one active site where histidine is modified. Correlation of residual activity with the number of histidines modified, as determined by absorbance at 240 nm, indicated that a maximum of three histidines are modified per subunit, two of which are essential for full inactivation. Comparison of the M. thermophila acetate kinase sequence with 56 putative acetate kinase sequences revealed eight highly conserved histidines, three of which (His-123, His-180, and His-208) are perfectly conserved. Diethylpyrocarbonate inactivation of the eight histidine 3 alanine variants indicated that His-180 and His-123 are in the active site and that the modification of both is necessary for full inactivation. Kinetic analyses of the eight variants showed that no other histidines are important for activity. Analysis of additional His-180 variants indicated that phosphorylation of His-180 is not essential for catalysis. Possible functions of His-180 are discussed.
Journal of Bacteriology, 2010
Short- and medium-chain acyl coenzyme A (acyl-CoA) synthetases catalyze the formation of acyl-CoA... more Short- and medium-chain acyl coenzyme A (acyl-CoA) synthetases catalyze the formation of acyl-CoA from an acyl substrate, ATP, and CoA. These enzymes catalyze mechanistically similar two-step reactions that proceed through an enzyme-bound acyl-AMP intermediate. Here we describe the characterization of a member of this enzyme family from the methane-producing archaeon Methanosarcina acetivorans . This enzyme, a medium-chain acyl-CoA synthetase designated Macs Ma , utilizes 2-methylbutyrate as its preferred substrate for acyl-CoA synthesis but cannot utilize acetate and thus cannot catalyze the first step of acetoclastic methanogenesis in M. acetivorans . When propionate or other less favorable acyl substrates, such as butyrate, 2-methylpropionate, or 2-methylvalerate, were utilized, the acyl-CoA was not produced or was produced at reduced levels. Instead, acyl-AMP and PP i were released in the absence of CoA, whereas in the presence of CoA, the intermediate was broken down into AMP a...
Journal of Bacteriology, 2001
Acetate kinase, an enzyme widely distributed in theBacteria and Archaea domains, catalyzes the ph... more Acetate kinase, an enzyme widely distributed in theBacteria and Archaea domains, catalyzes the phosphorylation of acetate. We have determined the three-dimensional structure of Methanosarcina thermophila acetate kinase bound to ADP through crystallography. As we previously predicted, acetate kinase contains a core fold that is topologically identical to that of the ADP-binding domains of glycerol kinase, hexokinase, the 70-kDa heat shock cognate (Hsc70), and actin. Numerous charged active-site residues are conserved within acetate kinases, but few are conserved within the phosphotransferase superfamily. The identity of the points of insertion of polypeptide segments into the core fold of the superfamily members indicates that the insertions existed in the common ancestor of the phosphotransferases. Another remarkable shared feature is the unusual, epsilon conformation of the residue that directly precedes a conserved glycine residue (Gly-331 in acetate kinase) that binds the α-phosp...
Journal of Bacteriology, 2011
The genome sequence of the aceticlastic methanoarchaeon Methanosaeta concilii GP6, comprised of a... more The genome sequence of the aceticlastic methanoarchaeon Methanosaeta concilii GP6, comprised of a 3,008,626-bp chromosome and an 18,019-bp episome, has been determined and exhibits considerable differences in gene content from that of Methanosaeta thermophila.
Genome Research, 2002
Methanogenesis, the biological production of methane, plays a pivotal role in the global carbon c... more Methanogenesis, the biological production of methane, plays a pivotal role in the global carbon cycle and contributes significantly to global warming. The majority of methane in nature is derived from acetate. Here we report the complete genome sequence of an acetate-utilizing methanogen, Methanosarcina acetivorans C2A. Methanosarcineae are the most metabolically diverse methanogens, thrive in a broad range of environments, and are unique among the Archaea in forming complex multicellular structures. This diversity is reflected in the genome ofM. acetivorans. At 5,751,492 base pairs it is by far the largest known archaeal genome. The 4524 open reading frames code for a strikingly wide and unanticipated variety of metabolic and cellular capabilities. The presence of novel methyltransferases indicates the likelihood of undiscovered natural energy sources for methanogenesis, whereas the presence of single-subunit carbon monoxide dehydrogenases raises the possibility of nonmethanogenic ...
Eukaryotic Cell, 2014
Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), previously thought to be present... more Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), previously thought to be present only in bacteria but recently found in fungi, catalyzes the formation of acetyl phosphate from xylulose 5-phosphate or fructose 6-phosphate. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Xfp, from the opportunistic fungal pathogen Cryptococcus neoformans , which has two XFP genes (designated XFP1 and XFP2 ). Our kinetic characterization of C. neoformans Xfp2 indicated the existence of both substrate cooperativity for all three substrates and allosteric regulation through the binding of effector molecules at sites separate from the active site. Prior to this study, Xfp enzymes from two bacterial genera had been characterized and were determined to follow Michaelis-Menten kinetics. C. neoformans Xfp2 is inhibited by ATP, phosphoenolpyruvate (PEP), and oxaloacetic acid (OAA) and activated by AMP. ATP is the strongest inhibitor, with a half-maximal inh...
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Papers by Cheryl Ingram-Smith