Requires Mg2+. All polyketide synthases, fatty-acid synthases and non-ribosomal peptide synthases require post-translational modification of their constituent acyl-carrier-protein (ACP) domains to become catalytically active. The inactive apo-proteins are converted into their active holo-forms by transfer of the 4'-phosphopantetheinyl moiety of CoA to the sidechain hydroxy group of a conserved serine residue in each ACP domain . The enzyme from human can activate both the ACP domain of the human cytosolic multifunctional fatty-acid synthase system (EC 2.3.1.85) and that associated with human mitochondria as well as peptidyl-carrier and acyl-carrier-proteins from prokaryotes . Removal of the 4-phosphopantetheinyl moiety from holo-ACP is carried out by EC 3.1.4.14, [acyl-carrier-protein] phosphodiesterase.
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
CoA-[4'-phosphopantetheine] + an apo-[acyl-carrier protein] = adenosine 3',5'-bisphosphate + an [acyl-carrier protein]
sequential binding mechanism: initial CoA- and Mg2+-binding followed by binding of acyl-carrier protein (ACP), nucleophilic attack of ACP-serine-hydroxylate on beta-phosphate of CoA followed by charge migration, Lys185-protonation, diphosphate-cleavage, and product dissociation, rate limiting step: release of 3',5'-ADP, key acid/base catalysts: residues E181 (CoA-binding) and K185 (Mg2+-binding)
Requires Mg2+. All polyketide synthases, fatty-acid synthases and non-ribosomal peptide synthases require post-translational modification of their constituent acyl-carrier-protein (ACP) domains to become catalytically active. The inactive apo-proteins are converted into their active holo-forms by transfer of the 4'-phosphopantetheinyl moiety of CoA to the sidechain hydroxy group of a conserved serine residue in each ACP domain [3]. The enzyme from human can activate both the ACP domain of the human cytosolic multifunctional fatty-acid synthase system (EC 2.3.1.85) and that associated with human mitochondria as well as peptidyl-carrier and acyl-carrier-proteins from prokaryotes [6]. Removal of the 4-phosphopantetheinyl moiety from holo-ACP is carried out by EC 3.1.4.14, [acyl-carrier-protein] phosphodiesterase.
single broad specificity enzyme for all posttranslational 4'-phosphopantetheinylation reactions, also capable of phosphopantetheinylation of peptidyl carrier and acyl carrier proteins from prokaryotes
development of a direct and continuous assay for this enzyme class based upon monitoring polarization of a fluorescent phosphopantetheine analogue as it is transferred from a low molecular weight coenzyme A substrate to higher molecular weight protein acceptor, utility of the method for the biochemical characterization of phosphopantetheinyl transferase Sfp, a canonical enzyme, recombinant enzyme with substrates VibB and 90 amino acid ACP (hACP) from human fatty acid synthase, overview
development of a direct and continuous assay for this enzyme class based upon monitoring polarization of a fluorescent phosphopantetheine analogue as it is transferred from a low molecular weight coenzyme A substrate to higher molecular weight protein acceptor, utility of the method for the biochemical characterization of phosphopantetheinyl transferase Sfp, a canonical enzyme, recombinant enzyme with substrates VibB and 90 amino acid ACP (hACP) from human fatty acid synthase, overview
AcpM, the meromycolate extension acyl carrier protein of Mycobacterium tuberculosis, is activated by the 4'-phosphopantetheinyl transferase PptT, a potential target of the multistep mycolic acid biosynthesis.
Crystal structure of the essential Mycobacterium tuberculosis phosphopantetheinyl transferase PptT, solved as a fusion protein with maltose binding protein.
Detection of soluble co-factor dependent protein expression in vivo: Application to the 4'-phosphopantetheinyl transferase PptT from Mycobacterium tuberculosis.
Inhibition of Indigoidine Synthesis as a High-Throughput Colourimetric Screen for Antibiotics Targeting the Essential Mycobacterium tuberculosis Phosphopantetheinyl Transferase PptT.
Insights from the docking and molecular dynamics simulation of the Phosphopantetheinyl transferase (PptT) structural model from Mycobacterium tuberculosis.
Structural and functional analysis of Rv3214 from Mycobacterium tuberculosis, a protein with conflicting functional annotations, leads to its characterization as a phosphatase.
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
apo-PPT (PDB: 2BYD) or in complex with coenzyme A (CoA, 5 mM) and Mg2+ (20 mM) (PDB: 2C43) or coenzyme A (2.5 mM) and acyl-carrier protein (S2156A mutant of ACP domain of fatty acid synthase) (PDB: 2CG5), precipitant: 14% PEG3350 and 0.05 M H3Cit/Na3Cit pH 5.7 or 2 M NaCl and 10% PEG6000 (complexes), apo-PPT: space group: P2(1)2(1)2(1), unit cell parameters: a: 63.78, b: 69.95, c: 71.24, alpha/beta fold with pseudo 2fold symmetry, N-terminal beta sheet (residues 91-116) connected to C-terminal beta sheet (residues 207-239) by a one residue linker and unique N-terminal and C-terminal extensions of 13 and 52 amino acids, respectively, PPT-CoA complex: space group: P2(1)2(1)2(1), unit cell parameters: a: 65.59, b: 68.96, c: 70.75, CoA-binding at the interface of N- and C-terminal domain mediated by PPT residues 47, 86, 110, 111, 185 (hydrophobic interactions, hydrogen bonds and salt bridges), and independent of Mg2+, Mg2+ bound through PPT residues 181 and 129 and coordinated by a water molecule, PPT-CoA-ACP complex: space group: P3(2)21, unit cell parameters: a, b: 69.36, c: 184.7, ACP-binding in the cleft between N- and C-terminal domain causes their rotation and slight closure, and is facilitated predominantly by hydrophobic interactions with PPT residues 51-54, 191, 144-148, 173, and 177 and a few polar interactions, disorder of C-terminal coil (residues 290-305), lack of Mg2+
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
from bacterial lysate by immobilized metal affinity chromatography followed by gel filtration chromatography on Superdex200 HiLoad 26/60 column and concentration to 20 mg/ml or anion exchange chromatography and dialysis
insights in molecular architecture and reaction mechanism of group II PPTs in contrast to group I PPTs (bacterial) enable screening for antibacterial agents which specifically inhibit bacterial PPTs