85-61-0Relevant articles and documents
Reconstitution of the pyridoxal 5′-phosphate (PLP) dependent enzyme serine palmitoyltransferase (SPT) with pyridoxal reveals a crucial role for the phosphate during catalysis
Beattie, Ashley E.,Clarke, David J.,Wadsworth, John M.,Lowther, Jonathan,Sin, Ho-Lam,Campopiano, Dominic J.
, p. 7058 - 7060 (2013)
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.
Structural basis for the activity and substrate specificity of fluoroacetyl-CoA thioesterase FIK
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.
, p. 22495 - 22504 (2010)
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.
An evolutionarily conserved alternate metal ligand is important for activity in α-isopropylmalate synthase from Mycobacterium tuberculosis
Frantom, Patrick A.,Birman, Yuliya,Hays, Brittani N.,Casey, Ashley K.
, p. 1784 - 1789 (2014)
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.
Chemical and enzymatic synthesis of tritium labelled coenzymes
Sidorov,Zverkov,Shram,Lazurkina,Myasoedov
, p. 465 - 473 (2003)
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
In Situ Assembly of Choline Acetyltransferase Ligands by a Hydrothiolation Reaction Reveals Key Determinants for Inhibitor Design
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)
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.
Discovery and Biosynthesis of Bolagladins: Unusual Lipodepsipeptides from Burkholderia gladioli Clinical Isolates**
Challis, Gregory L.,Dashti, Yousef,Jian, Xinyun,Mahenthiralingam, Eshwar,Mullins, Alex J.,Nakou, Ioanna T.,Webster, Gordon
, p. 21553 - 21561 (2020/10/02)
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
Structural insights into the inhibition mechanism of human sterol O-acyltransferase 1 by a competitive inhibitor
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
, (2020/05/27)
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.
Formyltetrahydrofolate Decarbonylase Synthesizes the Active Site CO Ligand of O2-Tolerant [NiFe] Hydrogenase
Schulz, Anne-Christine,Frielingsdorf, Stefan,Pommerening, Phillip,Lauterbach, Lars,Bistoni, Giovanni,Neese, Frank,Oestreich, Martin,Lenz, Oliver
, p. 1457 - 1464 (2020/01/31)
[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.
An Efficient Chemoenzymatic Synthesis of Coenzyme A and Its Disulfide
Mouterde, Louis M. M.,Stewart, Jon D.
, p. 954 - 959 (2016/06/13)
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.
Enzymatic characterization and elucidation of the catalytic mechanism of a recombinant bovine glycine N-acyltransferase
Badenhorst, Christoffel P. S.,Jooste, Maritza,Van Dijk, Alberdina A.
experimental part, p. 346 - 352 (2012/06/30)
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
