scholarly journals Identification of Essential Glutamates in the Acetate Kinase from Methanosarcina thermophila

1998 ◽  
Vol 180 (5) ◽  
pp. 1129-1134 ◽  
Author(s):  
Kavita Singh-Wissmann ◽  
Cheryl Ingram-Smith ◽  
Rebecca D. Miles ◽  
James G. Ferry
Keyword(s):  
Active Site ◽  
Kinase Activity ◽  
Amino Acid Sequences ◽  
Acetate Kinase ◽  
Acetyl Phosphate ◽  
Methanosarcina Thermophila ◽  
High Identity ◽  
E Coli ◽  
Metal Affinity ◽  
Enzyme Intermediate

ABSTRACT Acetate kinase catalyzes the reversible phosphorylation of acetate (CH3COO− + ATP⇄CH3CO2PO3 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 theM. thermophila enzyme to determine if any are essential for catalysis. The histidine-tagged altered enzymes were produced inE. 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 Km values for acetate and ATP with only moderate decreases ink 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 toN-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 theKm 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.

scholarly journals Characterization of the Acetate Binding Pocket in the Methanosarcina thermophila Acetate Kinase

2005 ◽  
Vol 187 (7) ◽  
pp. 2386-2394 ◽  
Author(s):  
Cheryl Ingram-Smith ◽  
Andrea Gorrell ◽  
Sarah H. Lawrence ◽  
Prabha Iyer ◽  
Kerry Smith ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Binding Pocket ◽  
Acetate Kinase ◽  
Phosphoryl Group ◽  
Acetyl Phosphate ◽  
Hydrophobic Pocket ◽  
Methanosarcina Thermophila ◽  
Methyl Group ◽  
Substrate Binding Sites

ABSTRACT Acetate kinase catalyzes the reversible magnesium-dependent synthesis of acetyl phosphate by transfer of the ATP γ-phosphoryl group to acetate. Inspection of the crystal structure of the Methanosarcina thermophila enzyme containing only ADP revealed a solvent-accessible hydrophobic pocket formed by residues Val93, Leu122, Phe179, and Pro232 in the active site cleft, which identified a potential acetate binding site. The hypothesis that this was a binding site was further supported by alignment of all acetate kinase sequences available from databases, which showed strict conservation of all four residues, and the recent crystal structure of the M. thermophila enzyme with acetate bound in this pocket. Replacement of each residue in the pocket produced variants with Km values for acetate that were 7- to 26-fold greater than that of the wild type, and perturbations of this binding pocket also altered the specificity for longer-chain carboxylic acids and acetyl phosphate. The kinetic analyses of variants combined with structural modeling indicated that the pocket has roles in binding the methyl group of acetate, influencing substrate specificity, and orienting the carboxyl group. The kinetic analyses also indicated that binding of acetyl phosphate is more dependent on interactions of the phosphate group with an unidentified residue than on interactions between the methyl group and the hydrophobic pocket. The analyses also indicated that Phe179 is essential for catalysis, possibly for domain closure. Alignments of acetate kinase, propionate kinase, and butyrate kinase sequences obtained from databases suggested that these enzymes have similar catalytic mechanisms and carboxylic acid substrate binding sites.

scholarly journals Characterization of CamH from Methanosarcina thermophila, Founding Member of a Subclass of the γ Class of Carbonic Anhydrases

2009 ◽  
Vol 192 (5) ◽  
pp. 1353-1360 ◽  
Author(s):  
Sabrina A. Zimmerman ◽  
Jean-Francois Tomb ◽  
James G. Ferry
Keyword(s):  
Active Site ◽  
Carbonic Anhydrases ◽  
Methanosarcina Thermophila ◽  
E Coli ◽  
Founding Member ◽  
Supplemental Zinc ◽  
Intermolecular Proton Transfer ◽  
Ping Pong ◽  
Iron Form

ABSTRACT The homotrimeric enzyme Mt-Cam from Methanosarcina thermophila is the archetype of the γ class of carbonic anhydrases. A search of databases queried with Mt-Cam revealed that a majority of the homologs comprise a putative subclass (CamH) in which there is major conservation of all of the residues essential for the archetype Mt-Cam except Glu62 and an acidic loop containing the essential proton shuttle residue Glu84. The CamH homolog from M. thermophila (Mt-CamH) was overproduced in Escherichia coli and characterized to validate its activity and initiate an investigation of the CamH subclass. The Mt-CamH homotrimer purified from E. coli cultured with supplemental zinc (Zn-Mt-CamH) contained 0.71 zinc and 0.15 iron per monomer and had k cat and kcat /Km values that were substantially lower than those for the zinc form of Mt-Cam (Zn-Mt-Cam). Mt-CamH purified from E. coli cultured with supplemental iron (Fe-Mt-CamH) was also a trimer containing 0.15 iron per monomer and only a trace amount of zinc and had an effective k cat (k cat eff) value normalized for iron that was 6-fold less than that for the iron form of Mt-Cam, whereas the k cat/Km eff was similar to that for Fe-Mt-Cam. Addition of 50 mM imidazole to the assay buffer increased the k cat eff of Fe-Mt-CamH more than 4-fold. Fe-Mt-CamH lost activity when it was exposed to air or 3% H2O2, which supports the hypothesis that Fe2+ has a role in the active site. The k cat for Fe-Mt-CamH was dependent on the concentration of buffer in a way that indicates that it acts as a second substrate in a “ping-pong” mechanism accepting a proton. The k cat/Km was not dependent on the buffer, consistent with the mechanism for all carbonic anhydrases in which the interconversion of CO2 and HCO3 − is separate from intermolecular proton transfer.

scholarly journals The Role of Active Site Residues in ATP Binding and Catalysis in the Methanosarcina thermophila Acetate Kinase

Life ◽  
2015 ◽  
Vol 5 (1) ◽  
pp. 861-871 ◽  
Author(s):  
Cheryl Ingram-Smith ◽  
Jeffrey Wharton ◽  
Christian Reinholz ◽  
Tara Doucet ◽  
Rachel Hesler ◽  
...  
Keyword(s):  
Active Site ◽  
Acetate Kinase ◽  
Atp Binding ◽  
Active Site Residues ◽  
Methanosarcina Thermophila

scholarly journals IMP-43 and IMP-44 Metallo-β-Lactamases with Increased Carbapenemase Activities in Multidrug-Resistant Pseudomonas aeruginosa

2013 ◽  
Vol 57 (9) ◽  
pp. 4427-4432 ◽  
Author(s):  
Tatsuya Tada ◽  
Tohru Miyoshi-Akiyama ◽  
Kayo Shimada ◽  
Masahiro Shimojima ◽  
Teruo Kirikae
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Amino Acid Residue ◽  
Active Site ◽  
Multidrug Resistant ◽  
Amino Acid Sequences ◽  
Medical Settings ◽  
Content Type ◽  
E Coli ◽  
Catalytic Activities

ABSTRACTTwo novel IMP-type metallo-β-lactamase variants, IMP-43 and IMP-44, were identified in multidrug-resistantPseudomonas aeruginosaisolates obtained in medical settings in Japan. Analysis of their predicted amino acid sequences revealed that IMP-43 had an amino acid substitution (Val67Phe) compared with IMP-7 and that IMP-44 had two substitutions (Val67Phe and Phe87Ser) compared with IMP-11. The amino acid residue at position 67 is located at the end of a loop close to the active site, consisting of residues 60 to 66 in IMP-1, and the amino acid residue at position 87 forms a hydrophobic patch close to the active site with other amino acids. AnEscherichia colistrain expressingblaIMP-43was more resistant to doripenem and meropenem but not to imipenem than one expressingblaIMP-7. AnE. colistrain expressingblaIMP-44was more resistant to doripenem, imipenem and meropenem than one expressingblaIMP-11. IMP-43 had more efficient catalytic activities against all three carbapenems than IMP-7, indicating that the Val67Phe substitution contributed to increased catalytic activities against carbapenems. IMP-44 had more efficient catalytic activities against all carbapenems tested than IMP-11, as well as increased activities compared with IMP-43, indicating that both the Val67Phe and Phe87Ser substitutions contributed to increased catalytic activities against carbapenems.

The function ofackAandptagenes is necessary for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in recombinantpha+Escherichia coli

1995 ◽  
Vol 41 (13) ◽  
pp. 200-206 ◽  
Author(s):  
Ho Gun Rhie ◽  
Douglas Dennis
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Kinase Activity ◽  
Acetate Kinase ◽  
Multicopy Plasmid ◽  
Biosynthesis Pathway ◽  
Acetyl Coa ◽  
E Coli ◽  
Acetyl Coa Synthesis ◽  
Copolymer Poly

In Escherichia coli carrying the poly(3-hydroxyalkanoate) (PHA) biosynthesis pathway on a plasmid (pha+), the function of the ackA (acetate kinase) and pta (phosphotransacetylase) genes is necessary for efficient incorporation of 3-hydroxyvalerate (3-HV) into the copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)). Recombinant pha+E. coli fadR atoC(Con) strains possessing mutations in ackA, pta, or both ackA and pta exhibited substantially reduced levels of 3-HV formation. Conversely, the same strains carrying the ackA gene on a multicopy plasmid exhibited an increase in 3-HV formation concomitant with a large increase in acetate kinase activity. However, if the strain possessing the multicopy ackA+plasmid was mutant at the pta locus, it lost the ability to incorporate significant amounts of 3-HV into P(3HB-co-3HV). In addition to the ackA pta pathway, there is an inducible activity that can also mediate the incorporation of 3-HV into P(3HB-co-3HV). This pathway is repressed by glucose and is not normally operative in P(3HB-co-3HV) production in recombinant pha+E. coli strains that are grown using glucose as the major carbon source. It appears likely that this activity is due to an inducible acetyl-CoA synthetase that converts propionate to propionyl-CoA.Key words: polyhydroxyalkanoates, acetate kinase, phosphotransacetylase, acetyl-CoA synthesis, propionyl-CoA synthesis.

scholarly journals Novel Pyrophosphate-Forming Acetate Kinase from the Protist Entamoeba histolytica

2012 ◽  
Vol 11 (10) ◽  
pp. 1249-1256 ◽  
Author(s):  
Matthew L. Fowler ◽  
Cheryl Ingram-Smith ◽  
Kerry S. Smith
Keyword(s):  
Entamoeba Histolytica ◽  
Acetate Kinase ◽  
Phosphoryl Group ◽  
Phosphoryl Transfer ◽  
Acetyl Phosphate ◽  
Phosphate Binding ◽  
Methanosarcina Thermophila ◽  
Inorganic Pyrophosphate ◽  
Content Type ◽  
Cell Extracts

ABSTRACTAcetate kinase (ACK) catalyzes the reversible synthesis of acetyl phosphate by transfer of the γ-phosphate of ATP to acetate. Here we report the first biochemical and kinetic characterization of a eukaryotic ACK, that from the protistEntamoeba histolytica. Our characterization revealed that this protist ACK is the only known member of the ASKHA structural superfamily, which includes acetate kinase, hexokinase, and other sugar kinases, to utilize inorganic pyrophosphate (PPi)/inorganic phosphate (Pi) as the sole phosphoryl donor/acceptor. Detection of ACK activity inE. histolyticacell extracts in the direction of acetate/PPiformation but not in the direction of acetyl phosphate/Piformation suggests that the physiological direction of the reaction is toward acetate/PPiproduction. Kinetic parameters determined for each direction of the reaction are consistent with this observation. TheE. histolyticaPPi-forming ACK follows a sequential mechanism, supporting a direct in-line phosphoryl transfer mechanism as previously reported for the well-characterizedMethanosarcina thermophilaATP-dependent ACK. Characterizations of enzyme variants altered in the putative acetate/acetyl phosphate binding pocket suggested that acetyl phosphate binding is not mediated solely through a hydrophobic interaction but also through the phosphoryl group, as for theM. thermophilaACK. However, there are key differences in the roles of certain active site residues between the two enzymes. The absence of known ACK partner enzymes raises the possibility that ACK is part of a novel pathway inEntamoeba.

scholarly journals Identification of the active-site lysine residues of two biosynthetic 3-dehydroquinases

1991 ◽  
Vol 275 (1) ◽  
pp. 1-6 ◽  
Author(s):  
S Chaudhuri ◽  
K Duncan ◽  
L D Graham ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Schiff Base ◽  
Nucleotide Sequence ◽  
Active Site ◽  
Active Sites ◽  
Amino Acid Sequences ◽  
E Coli ◽  
Lysine Residues ◽  
Schiff Base Formation ◽  
Base Formation

The lysine residues involved in Schiff-base formation at the active sites of both the 3-dehydroquinase component of the pentafunctional arom enzyme of Neurospora crassa and of the monofunctional 3-dehydroquinase of Escherichia coli were labelled by treatment with 3-dehydroquinate in the presence of NaB3H4. Radioactive peptides were isolated by h.p.l.c. following digestion with CNBr (and in one case after further digestion with trypsin). The sequence established for the N. crassa peptide was ALQHGDVVKLVVGAR, and that for the E. coli peptide was QSFDADIPKIA. An amended nucleotide sequence for the E. coli gene (aroD) that encode 3-dehydroquinase is also presented, along with a revised alignment of the deduced amino acid sequences for the biosynthetic enzymes.

scholarly journals Characterization of a 12-Kilodalton Rhodanese Encoded byglpE of Escherichia coli and Its Interaction with Thioredoxin

2000 ◽  
Vol 182 (8) ◽  
pp. 2277-2284 ◽  
Author(s):  
W. Keith Ray ◽  
Gang Zeng ◽  
M. Benjamin Potters ◽  
Aqil M. Mansuri ◽  
Timothy J. Larson
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Amino Acid Sequences ◽  
E Coli ◽  
Active Site Cysteine ◽  
Clusters Of Orthologous Groups ◽  
Thioredoxin 1 ◽  
First Time

ABSTRACT Rhodaneses catalyze the transfer of the sulfane sulfur from thiosulfate or thiosulfonates to thiophilic acceptors such as cyanide and dithiols. In this work, we define for the first time the gene, and hence the amino acid sequence, of a 12-kDa rhodanese fromEscherichia coli. Well-characterized rhodaneses are comprised of two structurally similar ca. 15-kDa domains. Hence, it is thought that duplication of an ancestral rhodanese gene gave rise to the genes that encode the two-domain rhodaneses. The glpEgene, a member of the sn-glycerol 3-phosphate (glp) regulon of E. coli, encodes the 12-kDa rhodanese. As for other characterized rhodaneses, kinetic analysis revealed that catalysis by purified GlpE occurs by way of an enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine. TheKm s for SSO3 2− and CN− were 78 and 17 mM, respectively. The apparent molecular mass of GlpE under nondenaturing conditions was 22.5 kDa, indicating that GlpE functions as a dimer. GlpE exhibited ak cat of 230 s−1. Thioredoxin 1 from E. coli, a small multifunctional dithiol protein, served as a sulfur acceptor substrate for GlpE with an apparentKm of 34 μM when thiosulfate was near itsKm , suggesting that thioredoxin 1 or related dithiol proteins could be physiological substrates for sulfurtransferases. The overall degree of amino acid sequence identity between GlpE and the active-site domain of mammalian rhodaneses is limited (∼17%). This work is significant because it begins to reveal the variation in amino acid sequences present in the sulfurtransferases. GlpE is the first among the 41 proteins in COG0607 (rhodanese-related sulfurtransferases) of the database Clusters of Orthologous Groups of proteins (http://www.ncbi.nlm.nih.gov/COG/ ) for which sulfurtransferase activity has been confirmed.

scholarly journals Structural and Functional Studies Suggest a Catalytic Mechanism for the Phosphotransacetylase from Methanosarcina thermophila

2006 ◽  
Vol 188 (3) ◽  
pp. 1143-1154 ◽  
Author(s):  
Sarah H. Lawrence ◽  
Kelvin B. Luther ◽  
Hermann Schindelin ◽  
James G. Ferry
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Van Der Waals Interactions ◽  
Nucleophilic Attack ◽  
Titration Calorimetry ◽  
Acetyl Phosphate ◽  
Methanosarcina Thermophila ◽  
Functional Studies ◽  
Active Site Cleft

ABSTRACT Phosphotransacetylase (EC 2.3.1.8) catalyzes reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. Two crystal structures of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila in complex with the substrate CoA revealed one CoA (CoA1) bound in the proposed active site cleft and an additional CoA (CoA2) bound at the periphery of the cleft. The results of isothermal titration calorimetry experiments are described, and they support the hypothesis that there are distinct high-affinity (equilibrium dissociation constant [KD ], 20 μM) and low-affinity (KD , 2 mM) CoA binding sites. The crystal structures indicated that binding of CoA1 is mediated by a series of hydrogen bonds and extensive van der Waals interactions with the enzyme and that there are fewer of these interactions between CoA2 and the enzyme. Different conformations of the protein observed in the crystal structures suggest that domain movements which alter the geometry of the active site cleft may contribute to catalysis. Kinetic and calorimetric analyses of site-specific replacement variants indicated that there are catalytic roles for Ser309 and Arg310, which are proximal to the reactive sulfhydryl of CoA1. The reaction is hypothesized to proceed through base-catalyzed abstraction of the thiol proton of CoA by the adjacent and invariant residue Asp316, followed by nucleophilic attack of the thiolate anion of CoA on the carbonyl carbon of acetyl phosphate. We propose that Arg310 binds acetyl phosphate and orients it for optimal nucleophilic attack. The hypothesized mechanism proceeds through a negatively charged transition state stabilized by hydrogen bond donation from Ser309.

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