- Reconstitution of the pyridoxal 5′-phosphate (PLP) dependent enzyme serine palmitoyltransferase (SPT) with pyridoxal reveals a crucial role for the phosphate during catalysis
-
The pyridoxal 5′-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) is required for de novo sphingolipid biosynthesis. A previous study revealed a novel and unexpected interaction between the hydroxyl group of the l-serine substrate and the 5′-phosphate group of PLP. By using pyridoxal (PL), the dephosphorylated analogue of vitamin B6, we show here that this interaction is important for substrate specificity and optimal catalytic efficiency.
- Beattie, Ashley E.,Clarke, David J.,Wadsworth, John M.,Lowther, Jonathan,Sin, Ho-Lam,Campopiano, Dominic J.
-
-
Read Online
- Structural basis for the activity and substrate specificity of fluoroacetyl-CoA thioesterase FIK
-
The thioesterase FlK from the fluoroacetate-producing Streptomyces cattleya catalyzes the hydrolysis of fluoroacetyl-coenzyme A. This provides an effective self-defense mechanism, preventing any fluoroacetyl-coenzyme A formed from being further metabolized to 4-hydroxy-trans-aconitate, a lethal inhibitor of the tricarboxylic acid cycle. Remarkably, FlK does not accept acetyl-coenzyme A as a substrate. Crystal structure analysis shows that FlK forms a dimer, in which each subunit adopts a hot dog fold as observed for type II thioesterases. Unlike other type II thioesterases, which invariably utilize either an aspartate or a glutamate as catalytic base, we show by site-directed mutagenesis and crystallography that FlK employs a catalytic triad composed of Thr42, His76, and a water molecule, analogous to the Ser/Cys-His-acid triad of type I thioesterases. Structural comparison of FlK complexed with various substrate analogues suggests that the interaction between the fluorine of the substrate and the side chain of Arg120 located opposite to the catalytic triad is essential for correct coordination of the substrate at the active site and therefore accounts for the substrate specificity.
- Dias, Marcio V. B.,Huang, Fanglu,Chirgadze, Dimitri Y.,Tosin, Manuela,Spiteller, Dieter,Dry, Emily F. V.,Leadlay, Peter F.,Spencer, Jonathan B.,Blundell, Tom L.
-
-
Read Online
- An evolutionarily conserved alternate metal ligand is important for activity in α-isopropylmalate synthase from Mycobacterium tuberculosis
-
Members of the DRE-TIM metallolyase superfamily rely on an active-site divalent cation to catalyze various reactions involving the making and breaking of carbon-carbon bonds. While the identity of the metal varies, the binding site is well-conserved at the superfamily level with an aspartic acid and two histidine residues acting as ligands to the metal. Previous structural and bioinformatics results indicate that the metal can adopt an alternate architecture through the addition of an asparagine residue as a fourth ligand. This asparagine residue is strictly conserved in all members of the DRE-TIM metallolyase superfamily except fungal homocitrate synthase (HCS-lys) where it is replaced with isoleucine. The role of this additional metal ligand in α-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) has been investigated using site-directed mutagenesis. Substitution of the asparagine ligand with alanine or isoleucine results in inactive enzymes with respect to α-isopropylmalate formation. Control experiments suggest that the substitutions have not drastically affected the enzyme's structure indicating that the asparagine residue is essential for catalysis. Interestingly, all enzyme variants retained acetyl CoA hydrolysis activity in the absence of α-ketoisovalerate, similar to the wild-type enzyme. In contrast to the requirement of magnesium for α-isopropylmalate formation, hydrolytic activity could be inhibited by the addition of magnesium chloride in wild-type, D81E, and N321A MtIPMS, but not in the other variants studied. Attempts to rescue loss of activity in N321I MtIPMS by mimicking the fungal HCS active site through the D81E/N321I double variant were unsuccessful. This suggests epistatic constraints in evolution of function in IPMS and HCS-lys enzymes.
- Frantom, Patrick A.,Birman, Yuliya,Hays, Brittani N.,Casey, Ashley K.
-
-
Read Online
- Chemical and enzymatic synthesis of tritium labelled coenzymes
-
Details of the synthesis of tritium labelled co enzymes-nicotinamide adenine dinucleotide and coenzyme A by isotope exchange and enzymatic reactions are reported. It was established, that among the investigated chemical reactions, the most effective is solid state isotope exchange with gaseous tritium. This method was used to produce [3H] nicotinamide adenine dinucleotide (111 Ci/ mmol), [3H] coenzyme A (3.9 Ci/mmol) and D-[G-3H] pantothenic acid (43 Ci/ mmol). It was shown that most of the tritium in the labeled nicotinamide adenine dinucleotide and coenzyme A was localized in the nicotinamide (98%) and adenine (89%) sites, respectively. For synthesis of coenzymes labelled with tritium at other sites we developed enzymatic methods which used labelled precursors. Optimum conditions for enzymatic synthesis of [adenine-3H] nicotinamide adenine dinucleotide from [2,8-3H] ATP and [pantothenate-3H] coenzyme A from D-[G-3H] pantothenic acid were determined. The tritium labelled acetyl coenzyme A was synthesized by acetylation of labelled coenzyme A with acetic anhydride. The methods chosen allow one to produce tritium labelled coenzymes at high specific activity. Copyright
- Sidorov,Zverkov,Shram,Lazurkina,Myasoedov
-
-
Read Online
- In Situ Assembly of Choline Acetyltransferase Ligands by a Hydrothiolation Reaction Reveals Key Determinants for Inhibitor Design
-
The potential drug target choline acetyltransferase (ChAT) catalyses the production of the neurotransmitter acetylcholine in cholinergic neurons, T-cells, and B-cells. Herein, we show that arylvinylpyridiniums (AVPs), the most widely studied class of ChAT inhibitors, act as substrate in an unusual coenzyme A-dependent hydrothiolation reaction. This in situ synthesis yields an adduct that is the actual enzyme inhibitor. The adduct is deeply buried in the active site tunnel of ChAT and interactions with a hydrophobic pocket near the choline binding site have major implications for the molecular recognition of inhibitors. Our findings clarify the inhibition mechanism of AVPs, establish a drug modality that exploits a target-catalysed reaction between exogenous and endogenous precursors, and provide new directions for the development of ChAT inhibitors with improved potency and bioactivity.
- Wiktelius, Daniel,Allgardsson, Anders,Bergstr?m, Tomas,Hoster, Norman,Akfur, Christine,Forsgren, Nina,Lejon, Christian,Hedenstr?m, Mattias,Linusson, Anna,Ekstr?m, Fredrik
-
supporting information
p. 813 - 819
(2020/12/09)
-
- Discovery and Biosynthesis of Bolagladins: Unusual Lipodepsipeptides from Burkholderia gladioli Clinical Isolates**
-
Two Burkholderia gladioli strains isolated from the lungs of cystic fibrosis patients were found to produce unusual lipodepsipeptides containing a unique citrate-derived fatty acid and a rare dehydro-β-alanine residue. The gene cluster responsible for the
- Challis, Gregory L.,Dashti, Yousef,Jian, Xinyun,Mahenthiralingam, Eshwar,Mullins, Alex J.,Nakou, Ioanna T.,Webster, Gordon
-
p. 21553 - 21561
(2020/10/02)
-
- Structural insights into the inhibition mechanism of human sterol O-acyltransferase 1 by a competitive inhibitor
-
Sterol O-acyltransferase 1 (SOAT1) is an endoplasmic reticulum (ER) resident, multi-transmembrane enzyme that belongs to the membrane-bound O-acyltransferase (MBOAT) family. It catalyzes the esterification of cholesterol to generate cholesteryl esters for cholesterol storage. SOAT1 is a target to treat several human diseases. However, its structure and mechanism remain elusive since its discovery. Here, we report the structure of human SOAT1 (hSOAT1) determined by cryo-EM. hSOAT1 is a tetramer consisted of a dimer of dimer. The structure of hSOAT1 dimer at 3.5 ? resolution reveals that a small molecule inhibitor CI-976 binds inside the catalytic chamber and blocks the accessibility of the active site residues H460, N421 and W420. Our results pave the way for future mechanistic study and rational drug design targeting hSOAT1 and other mammalian MBOAT family members.
- Chang, Catherine C. Y.,Chang, Ta-Yuan,Chen, Lei,Chen, Si-Cong,Guan, Chengcheng,Kang, Yunlu,Luo, Tuoping,Nishi, Koji,Niu, Yange,Wu, Jing-Xiang
-
-
- Formyltetrahydrofolate Decarbonylase Synthesizes the Active Site CO Ligand of O2-Tolerant [NiFe] Hydrogenase
-
[NiFe] hydrogenases catalyze the reversible oxidation of molecular hydrogen into two protons and two electrons. A key organometallic chemistry feature of the NiFe active site is that the iron atom is co-coordinated by two cyanides (CN-) and one carbon monoxide (CO) ligand. Biosynthesis of the NiFe(CN)2(CO) cofactor requires the activity of at least six maturation proteins, designated HypA-F. An additional maturase, HypX, is required for CO ligand synthesis under aerobic conditions, and preliminary in vivo data indicated that HypX releases CO using N10-formyltetrahydrofolate (N10-formyl-THF) as the substrate. HypX has a bipartite structure composed of an N-terminal module similar to N10-formyl-THF transferases and a C-terminal module homologous to enoyl-CoA hydratases/isomerases. This composition suggested that CO production takes place in two consecutive reactions. Here, we present in vitro evidence that purified HypX first transfers the formyl group of N10-formyl-THF to produce formyl-coenzyme A (formyl-CoA) as a central reaction intermediate. In a second step, formyl-CoA is decarbonylated, resulting in free CoA and carbon monoxide. Purified HypX proved to be metal-free, which makes it a unique catalyst among the group of CO-releasing enzymes.
- Schulz, Anne-Christine,Frielingsdorf, Stefan,Pommerening, Phillip,Lauterbach, Lars,Bistoni, Giovanni,Neese, Frank,Oestreich, Martin,Lenz, Oliver
-
p. 1457 - 1464
(2020/01/31)
-
- An Efficient Chemoenzymatic Synthesis of Coenzyme A and Its Disulfide
-
We have developed a chemoenzymatic route to coenzyme A (CoASH) and its disulfide that is amenable to gram-scale synthesis using standard laboratory equipment. By synthesizing the symmetrical disulfide of pantetheine (pantethine), we avoided the need to mask the reactive sulfhydryl and also prevented sulfur oxidation byproducts. No chromatography is required in our synthetic route to pantethine, which facilitates scale-up. Furthermore, we discovered that all three enzymes of the CoASH salvage pathway (pantetheine kinase, phosphopantetheine adenyltransferase, and dephospho-coenzyme A kinase) accept the disulfide of the natural substrates and functionalize both ends of the molecules. This yields CoA disulfide as the product of the enzymatic cascade, a much more stable form of the cofactor. Free CoASH can be prepared by in situ S-S reduction.
- Mouterde, Louis M. M.,Stewart, Jon D.
-
p. 954 - 959
(2016/06/13)
-
- Enzymatic characterization and elucidation of the catalytic mechanism of a recombinant bovine glycine N-acyltransferase
-
Glycine conjugation, a phase II detoxification process, is catalyzed by glycine N-acyltransferase (GLYAT; E.C. 2.3.1.13). GLYAT detoxifies various xenobiotics, such as benzoic acid, and endogenous organic acids, such as isovaleric acid, which makes GLYAT important in the management of organic acidemias in humans. We cloned the open reading frame encoding the bovine ortholog of GLYAT from bovine liver mRNA into the bacterial expression vector pColdIII. The recombinant enzyme was expressed, partially purified, and enzymatically characterized. Protein modeling was used to predict Glu 226 of bovine GLYAT to be catalytically important. This was assessed by constructing an E226Q mutant and comparing its enzyme kinetics to that of the wild-type recombinant bovine GLYAT. The Michaelis constants for benzoyl-CoA and glycine were determined and were similar for wild-type recombinant GLYAT, E226Q recombinant GLYAT, and GLYAT present in bovine liver. At pH 8.0, the E226Q mutant GLYAT had decreased activity, which could be compensated for by increasing the reaction pH. This suggested a catalytic mechanism in which Glu226 functions to deprotonate glycine, facilitating nucleophilic attack on the acyl- CoA. The recombinant bovine GLYAT enzyme, combined with this new understanding of its active site and reaction mechanism, could be a powerful tool to investigate the functional significance of GLYAT sequence variations. Eventually, this should facilitate investigations into the impact of known and novel sequence variations in the human GLYAT gene. Copyright
- Badenhorst, Christoffel P. S.,Jooste, Maritza,Van Dijk, Alberdina A.
-
experimental part
p. 346 - 352
(2012/06/30)
-
- Functional characterization of an NADPH dependent 2-alkyl-3-ketoalkanoic acid reductase involved in olefin biosynthesis in stenotrophomonas maltophilia
-
OleD is shown to play a key reductive role in the generation of alkenes (olefins) from acyl thioesters in Stenotrophomonas maltophilia. The gene coding for OleD clusters with three other genes, oleABC, and all appear to be transcribed in the same direction as an operon in various olefin producing bacteria. In this study, a series of substrates varying in chain length and stereochemistry were synthesized and used to elucidate the functional role and substrate specificity of OleD. We demonstrated that OleD, which is an NADP(H) dependent reductase, is a homodimer which catalyzes the reversible stereospecific reduction of 2-alkyl-3-ketoalkanoic acids. Maximal catalytic efficiency was observed with syn-2-decyl-3-hydroxytetradecanoic acid, with a kcat/Km 5- and 8-fold higher than for syn-2-octyl-3- hydroxydodecanoic acid and syn-2-hexyl-3-hydroxydecanoic acid, respectively. OleD activity was not observed with syn-2-butyl-3-hydroxyoctanoic acid and compounds lacking a 2-alkyl group such as 3-ketodecanoic and 3-hydroxydecanoic acids, suggesting the necessity of the 2-alkyl chain for enzyme recognition and catalysis. Using diastereomeric pairs of substrates and 4 enantiopure isomers of 2-hexyl-3-hydroxydecanoic acid of known stereochemistry, OleD was shown to have a marked stereochemical preference for the (2R,3S)-isomer. Finally, experiments involving OleA and OleD demonstrate the first 3 steps and stereochemical course in olefin formation from acyl thioesters; condensation to form a 2-alkyl-3-ketoacyl thioester, subsequent thioester hydrolysis, and ketone reduction.
- Bonnett, Shilah A.,Papireddy, Kancharla,Higgins, Samuel,Del Cardayre, Stephen,Reynolds, Kevin A.
-
experimental part
p. 9633 - 9640
(2012/07/16)
-
- Activity of 3-ketosteroid 9α-hydroxylase (KshAB) indicates cholesterol side chain and ring degradation occur simultaneously in Mycobacterium tuberculosis
-
Mycobacterium tuberculosis (Mtb), a significant global pathogen, contains a cholesterol catabolic pathway. Although the precise role of cholesterol catabolism in Mtb remains unclear, the Rieske monooxygenase in this pathway, 3-ketosteroid 9α-hydroxylase (KshAB), has been identified as a virulence factor. To investigate the physiological substrate of KshAB, a rhodococcal acyl-CoA synthetase was used to produce the coenzyme A thioesters of two cholesterol derivatives: 3-oxo-23,24-bisnorchol-4- en-22-oic acid (forming 4-BNC-CoA) and 3-oxo-23,24-bisnorchola- 1,4-dien-22-oic acid (forming 1,4-BNC-CoA). The apparent specificity constant (kcat/Km) of KshAB for the CoA thioester substrates was 20-30 times that for the corresponding 17-keto compounds previously proposed as physiological substrates. The apparent KmO2 was 90 ± 10 μM in the presence of 1,4-BNC-CoA, consistent with the value for two other cholesterol catabolic oxygenases. The Δ1 ketosteroid dehydrogenase KstD acted with KshAB to cleave steroid ring B with a specific activity eight times greater for a CoA thioester than the corresponding ketone. Finally, modeling 1,4-BNC-CoA into the KshA crystal structure suggested that the CoA moiety binds in a pocket at the mouth of the active site channel and could contribute to substrate specificity. These results indicate that the physiological substrates of KshAB are CoA thioester intermediates of cholesterol side chain degradation and that side chain and ring degradation occur concurrently in Mtb. This finding has implications for steroid metabolites potentially released by the pathogen during infection and for the design of inhibitors for cholesterol-degrading enzymes. The methodologies and rhodococcal enzymes used to generate thioesters will facilitate the further study of cholesterol catabolism.
- Capyk, Jenna K.,Casabon, Israel,Gruninger, Robert,Strynadka, Natalie C.,Eltis, Lindsay D.
-
experimental part
p. 40717 - 40724
(2012/06/29)
-
- Serine 254 enhances an induced fit mechanism in murine 5-aminolevulinate synthase
-
5-Aminolevulinate synthase (EC 2.3.1.37) (ALAS), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyzes the initial step of heme biosynthesis in animals, fungi, and some bacteria. Condensation of glycine and succinyl coenzyme A produces 5-aminolevulinate, coenzyme A, and carbon dioxide. X-ray crystal structures of Rhodobacter capsulatus ALAS reveal that a conserved active site serine moves to within hydrogen bonding distance of the phenolic oxygen of the PLP cofactor in the closed substrate-bound enzyme conformation and within 3-4 Ae of the thioester sulfur atom of bound succinyl-CoA. To evaluate the role(s) of this residue in enzymatic activity, the equivalent serine in murine erythroid ALAS was substituted with alanine or threonine. Although both the KmSCoA and kcat values of the S254A variant increased, by 25- and 2-fold, respectively, the S254T substitution decreased kcat without altering Km SCoA. Furthermore, in relation to wild-type ALAS, the catalytic efficiency of S254A toward glycine improved ～3-fold, whereas that of S254T diminished ～3-fold. Circular dichroism spectroscopy revealed that removal of the side chain hydroxyl group in the S254A variant altered the microenvironment of the PLP cofactor and hindered succinyl-CoA binding. Transient kinetic analyses of the variant-catalyzed reactions and protein fluorescence quenching upon 5-aminolevulinate binding demonstrated that the protein conformational transition step associated with product release was predominantly affected. We propose the following: 1) Ser-254 is critical for formation of a competent catalytic complex by coupling succinyl-CoA binding to enzyme conformational equilibria, and 2) the role of the active site serine should be extended to the entire α-oxoamine synthase family of PLP-dependent enzymes.
- Lendrihas, Thomas,Hunter, Gregory A.,Ferreira, Gloria C.
-
experimental part
p. 3351 - 3359
(2011/02/26)
-
- Unprecedented acetoacetyl-coenzyme A synthesizing enzyme of the thiolase superfamily involved in the mevalonate pathway
-
Acetoacetyl-CoA is the precursor of 3-hydroxy-3-methylglutaryl (HMG)-CoA in the mevalonate pathway, which is essential for terpenoid backbone biosynthesis. Acetoacetyl-CoA is also the precursor of poly-β-hydroxybutyrate, a polymer belonging to the polyester class produced by microorganisms. The de novo synthesis of acetoacetyl-CoA is usually catalyzed by acetoacetyl-CoA thiolase via a thioester-dependent Claisen condensation reaction between two molecules of acetyl-CoA. Here, we report that nphT7, found in the mevalonate pathway gene cluster from a soil-isolated Streptomyces sp. strain, encodes an unusual acetoacetyl-CoA synthesizing enzyme. The recombinant enzyme overexpressed in Escherichia coli catalyzes a single condensation of acetyl-CoA and malonyl-CoA to give acetoacetyl-CoA and CoA. Replacement of malonyl-CoA with malonyl-(acyl carrier protein) resulted in loss of the condensation activity. No acetoacetyl-CoA synthesizing activity was detected through the condensation of two molecules of acetyl-CoA. Based on these properties of NphT7, we propose to name this unusual enzyme of the thiolase superfamily acetoacetyl-CoA synthase. Co-expression of nphT7 with the HMG-CoA synthase gene and the HMG-CoA reductase gene in a heterologous host allowed 3.5-fold higher production of mevalonate than when only the HMG-CoA synthase and HMG-CoA reductase genes were expressed. This result suggests that nphT7 can be used to significantly increase the concentration of acetoacetyl-CoA in cells, eventually leading to the production of useful terpenoids and poly-β-hydroxybutyrate.
- Okamura, Eiji,Tomita, Takeo,Sawa, Ryuichi,Nishiyama, Makoto,Kuzuyama, Tomohisa
-
experimental part
p. 11265 - 11270
(2011/01/04)
-
- Michael acceptor-containing coenzyme a analogues as inhibitors of the atypical coenzyme a disulfide reductase from staphylococcus aureus
-
Coenzyme A (CoA) analogues containing α,β-unsaturated ester, ketone, and sulfone moieties were prepared by chemo-enzymatic synthesis as inhibitors of coenzyme A disulfide reductase (CoADR), a proven and as yet unexploited drug target in Staphylococcus aureus. Among these Michael acceptor-containing CoA analogues, which were designed to target CoADR's single essential active site cysteine for conjugate addition, a phenyl vinyl sulfone-containing analogue showed the most potent inhibition with a competitive Ki of ～40 nM, and time-dependent inactivation with a second-order rate of inactivation constant of ～40 000 s -1?M-1. Our results suggest that electrophilic substrate analogues should be considered as potential inhibitors of other medicinally relevant disulfide reductase enzymes.
- Van Der Westhuyzen, Renier,Strauss, Erick
-
supporting information; experimental part
p. 12853 - 12855
(2010/11/05)
-
- A three enzyme pathway for 2-amino-3-hydroxycyclopent-2-enone formation and incorporation in natural product biosynthesis
-
A number of natural products contain a 2-amino-3-hydroxycyclopent-2-enone five membered ring, termed C5N, which is condensed via an amide linkage to a variety of polyketide-derived polyenoic acid scaffolds. Bacterial genome mining indicates three tandem ORFs that may be involved in C5N formation and subsequent installation in amide linkages. We show that the protein products of three tandem ORFs (ORF33-35) from the ECO-02301 biosynthetic gene cluster in Streptomyces aizunenesis NRRL-B-11277, when purified from Escherichia coli, demonstrate the requisite enzyme activities for C5N formation and amide ligation. First, succinyl-CoA and glycine are condensed to generate 5-aminolevulinate (ALA) by a dedicated PLP-dependent ALA synthase (ORF34). Then ALA is converted to ALA-CoA through an ALA-AMP intermediate by an acyl-CoA ligase (ORF35). ALA-CoA is unstable and has a half-life of ～10 min under incubation conditions for off-pathway cyclization to 2,5-piperidinedione. The ALA synthase can compete with the nonenzymatic decomposition route and act in a novel second transformation, cyclizing ALA-CoA to C5N. C 5N is then a substrate for the third enzyme, an ATP-dependent amide synthetase (ORF33). Using octatrienoic acid as a mimic of the C56 polyenoic acid scaffold of ECO-02301, formation of the octatrienyl-C 5N product was observed. This three enzyme pathway is likely the general route to the C5N ring system in other natural products, including the antibiotic moenomycin.
- Zhang, Wenjun,Bolla, Megan L.,Kahne, Daniel,Walsh, Christopher T.
-
experimental part
p. 6402 - 6411
(2010/07/04)
-
- Structure-activity analysis of base and enzyme-catalyzed 4-hydroxybenzoyl coenzyme A hydrolysis
-
In this study, the second-order rate constant k2 of base-catalyzed hydrolysis and the values of kcat, Km and kcat/Km of wild-type Pseudomonas sp. CBS3 4-hydroxybenzoyl coenzyme A (4-HBA-CoA) thioesterase-catalyzed hydrolysis of 4-HBA-CoA and its para-substituted analogs were measured. For the base-catalyzed hydrolysis, the plot of log k2 vs the σ value of the para-substituents was linear with a slope (ρ) of 1.5. In the case of the enzyme-catalyzed hydrolysis, the kcat/Km values measured for the para-substituted analogs defined substrate specificity. Asp32 was shown to play a key role in substrate recognition, and in particular, in the discrimination between the targeted substrate and other cellular benzoyl-CoA thioesters.
- Song, Feng,Zhuang, Zhihao,Dunaway-Mariano, Debra
-
-
- The first thermophilic α-oxoamine synthase family enzyme that has activities of 2-amino-3-ketobutyrate CoA ligase and 7-keto-8-aminopelargonic acid synthase: Cloning and overexpression of the gene from an extreme thermophile, Thermus thermophilus, and characterization of its gene product
-
The first thermophilic α-oxoamine synthase family enzyme was identified. The gene (ORF TTHA1582), which is annotated to code putative α-oxoamine synthase family enzymes, 7-keto-8-aminopelargonic acid (KAPA) synthase (BioF, 8-amino-7-oxononanoate synthase, EC 2.3.1.47) and 2-amino-3-ketobutyrate CoA ligase (KBL, EC 2.3.1.29), in a genomic database, was cloned from an extreme thermophile, Thermus thermophilus, and overexpressed in Escherichia coli. The recombinant TTHA1582 protein was purified and characterized. It exhibited activity of BioF, which catalyzes the condensation of pimeloyl-CoA and L-alanine to produce a biotin intermediate KAPA, CoASH, and CO2 with pyridoxal 5′-phosphate as a cofactor. The protein is a dimer with a subunit of 43 kDa that shows an amino acid sequence identity of 35% with E. coli BioF. The optimum temperature and pH were about 70 °C and about 6.0. The enzyme showed high thermostability at temperatures of up to 70 °C for 1 h, and a half-life of 1 h at 80 °C. Thus the TTHA1582 protein was found to have the highest optimum temperature and thermostablility of the α-oxoamine synthase family enzymes so far reported. Substrate specificity experiments revealed that it was also able to catalyze the KBL reaction, which used acetyl-CoA and glycine as substrates, and that enzyme activity was seen with the following combinations of substrates: acetyl-CoA and glycine, L-alanine, or L-serine; pimeloyl-CoA and L-alanine, glycine, or L-serine; palmitoyl-CoA and L-alanine. This suggests that the recombinant TTHA1582 protein has broad substrate specificity, unlike the reported mesophilic enzymes of the α-oxoamine synthase family.
- Kubota, Takaaki,Shimono, Jyunpei,Kanameda, Chie,Izumi, Yoshikazu
-
p. 3033 - 3040
(2008/03/15)
-
- Hepatic enzymatic synthesis and hydrolysis of CoA esters of solvent-derived oxa acids
-
Many ethylene glycol-derived solvents are oxidized to xenobiotic alkoxyacetic acids (3-oxa acids) by hepatic enzymes. The toxicity of these ubiquitous solvents has been associated with their oxa acid metabolites. For many xenobiotic carboxylic acids, the toxicity is associated with the CoA ester of the acid. In this study, related alkoxyacetic acids were evaluated as potential substrates for acyl-CoA synthetases found in mitochondrial, peroxisomal, and microsomal fractions isolated from rat liver. Likewise, chemically synthesized oxa acyl-CoAs were used as substrates for acyl-CoA hydrolases associated with the same rat liver fractions. Activities of the xenobiotic oxygen-substituted substrates were compared with analogous physiologic aliphatic substrates by UV-vis spectrophotometric methods. All of the solvent-derived oxa acids were reasonable substrates for the acyl-CoA synthetases, although their activity was usually less than the corresponding physiologic acid. Acyl-CoA hydrolase activities were decreased compared with acyl-CoA synthetase activities for all substrates, especially for the oxa acyl-CoAs. These studies suggest that these xenobiotic carboxylic acids may be converted to reactive acyl-CoA moieties which will persist in areas of the cell proximal to lipid synthesis, β-oxidation, protein acylation, and amino acid conjugation. The interaction of these xenobiotic acyl-CoAs with those processes may be important to their toxicity and/or detoxification.
- Panuganti, Sree D.,Penn, Jill M.,Moore, Kathleen H.
-
-
- Coenzyme A hemithioacetals as easily prepared inhibitors of CoA ester-utilizing enzymes
-
Hemithioacetals are formed by reactions of coenzyme A (CoA) with aldehydes in aqueous solution. Equilibria for hemithioacetal formation with four commercially available aldehydes and rate constants for hemithioacetal dissociation have been studied. The hemithioacetals are viewed as acyl-CoA analogs having a tetrahedral center in place of the planar trigonal thioester carbonyl carbon. These compounds may serve as mimics of the tetrahedral intermediate or transition state in the reactions of acyl-CoA dependent acyltransferase enzymes. The hemithioacetal generated by reaction of CoA with formaldehyde is a poor inhibitor of chloramphenicol acetyltransferase, with a K(i) more than 6-fold higher than the K(m) for the substrate acetyl-CoA. The hemithioacetals formed by reaction of CoA with acetaldehyde and trifluroacetaldehyde are substantially better inhibitors, with K(i) values approximately 2.4-fold and 10-fold lower than the K(m) values for acetyl-CoA, respectively. The hemithioacetal formed by reaction of CoA with succinic semialdehyde inhibits succinic thiokinase, with a K(i) 4-fold lower than the K(m) for the substrate succinyl-CoA. The CoA hemithioacetals provide a novel readily accessible new class of acyl-CoA analogs for use in mechanistic and structural studies of CoA ester-utilizing enzymes.
- Schwartz, Benjamin,Vogel, Kurt W.,Drueckhammer, Dale G.
-
p. 9356 - 9361
(2007/10/03)
-
- Kinetics and Equilibria of Thiol/Disulfide Interchange Reactions of Selected Biological Thiols and Related Molecules with Oxidized Glutathione
-
Rate constants for reaction of coenzyme A and cysteine with oxidized glutathione (GSSG) and equilibrium constants for the reaction of coenzyme A, cysteine, homocysteine, cysteamine, and related thiols with GSSG by thiol/disulfide interchange were determined over a range of pD values by NMR spectroscopy.The rate constants for reaction of the thiolate anion forms of coenzyme A and cysteine with GSSG suggest that reduction of GSSG by coenzyme A and cysteine is a mechanistically uncomplicated SN2 reaction.Equilibrium constants for the thiol/disulfide interchange reactions show a strong dependence on the Bronsted basicity of the thiolate anion.In a similar way, ΔE0', the difference between the half-cell potentials for the RSSR/RSH and GSSG/GSH redox couples, is linearly dependent on the difference between the pKA values of RSH and glutathione: ΔE0' = 64ΔpKA - 7.7 where ΔE0' is in units of mV.The reducing strength at a given pH is also determined by the fraction of the thiol present in the reactive thiolate form.At pD 7, the half-cell potentials for coenzyme A, cysteine, homocysteine, and cysteamine are close to that of glutathione, the major intracellular thiol redox system, which suggests that small changes in the intracellular redox potential can cause significant changes in the intracellular distribution of these biological thiols between their reduced and oxidized forms.
- Keire, David A.,Strauss, Erin,Guo, Wei,Noszal, Bela,Rabenstein, Dallas L.
-
p. 123 - 127
(2007/10/02)
-
- Leaving Group Effects in Thiolester Alkaline Hydrolysis. Part 1. A Keten-mediated (E1cB) Pathway for Basic Hydrolysis of S-Acetoacetylcoenzyme A and Analogues
-
The basic hydrolysis of a series of leaving-group substituted acetothiolacetates (CH3COCH2COSR) has been studied in aqueous media.Hydrolysis of N-acetyl-S-acetoacetylcysteinamine follows a kinetic ionisation curve with an inflexion corresponding to the pK of this ester as determined by spectrophotometric and electrometric titrations.The rate constant at high pH was shown to follow a Broensted relationship with βL.G. -1.13, where βL.G. is the slope of a plot of the logarithm of the rate constant versus the pKa of the conjugate acid of the leaving group.This, and other evidence from rate comparisons, activation parameters, and kinetic solvent isotope effects, indicated an E1cB hydrolytic mechanism involving unimolecular collapse of the ester enolate ions via a ketenoid transition-state.S-Acetoacetylcoenzyme-A was also hydrolysed in base by this mechanism.Direct comparison of rates of leaving group expulsion for ArS and ArO was possible by means of this unimolecular process.For a leaving group with pKL.G. 10, the oxyanion departs ca. 1 or 2 orders of magnitude faster than the thiolate anion; for pKL.G. 6.0, the advantage of oxygen over sulphur is 103-104 fold.In a direct structural comparison, PhS departs 32 times as rapidly as PhO.The contribution of steric release in the E1cB transition-state for S-t-butyl acetothiolacetate hydrolysis is discussed.The pKa values of some acetothiolacetates were measured.
- Douglas, Kenneth T.,Yaggi, Norbert F.
-
p. 1037 - 1044
(2007/10/02)
-
- Coenzyme A: pKa and γ Values
-
The pKa and γ values at 25 deg C, I 0.1 M, of the mercapto group of coenzyme A, were calculated to be 10.35+/-0.15 and 0.70+/-0.02 respectively.The γ value was calculated from differences in the parition coefficient of quinazoline between cyclohexane and aqueous buffers and that between cyclohexane and aqueous buffers which contained coenzyme A.The described procedure could be used to obtain γ values of other nucleophilic reagents which, like coenzyme A, absorb u.v. energy strongly.
- Pitman, Ian H.,Morris, Ian J.
-
p. 1625 - 1630
(2007/10/02)
-
