Application | Comment | Organism |
---|---|---|
molecular biology | 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 | Homo sapiens |
Cloned (Comment) | Organism |
---|---|
in pCOEX1 for expression with TEV protease-cleavable N-terminal hexa-His-tag in Escherichia coli BL21(DE3)-R3 | Homo sapiens |
Crystallization (Comment) | Organism |
---|---|
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+ | Homo sapiens |
Protein Variants | Comment | Organism |
---|---|---|
D129A | reduced Mg2+ affinity and catalytic efficiency, D129 plays a role in Mg2+-coordination | Homo sapiens |
E181A | significant loss in enzyme activity, reduced Mg2+ affinity and catalytic efficiency | Homo sapiens |
E181Q | significant loss in enzyme activity, reduced Mg2+ affinity (20fold) and catalytic efficiency (300fold) | Homo sapiens |
K185 | significant loss in enzyme activity, reduced catalytic efficiency | Homo sapiens |
Q112E | slightly reduced catalytic efficiency | Homo sapiens |
Q112E, E181Q | double mutant, reduced Mg2+ affinity (200fold) and catalytic efficiency | Homo sapiens |
R47A | reduced coenzymeA and Mg2+ affinity, increased catalytic efficiency | Homo sapiens |
R86A | reduced coenzymeA and Mg2+ affinity, increased catalytic efficiency | Homo sapiens |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
acetyl-CoA | double mutant Q112E, E181Q, Kcat/KM: 0.2 1/min/mM | Homo sapiens | |
additional information | - |
Mg2+ | mutant D129A, Kcat/KM: 0.0001 1/min/mM | Homo sapiens | |
additional information | - |
acetyl-CoA | mutant D129A, Kcat/KM: 0.04 1/min/mM | Homo sapiens | |
additional information | - |
Mg2+ | mutant E181A, Kcat/KM: 0.001 1/min/mM | Homo sapiens | |
additional information | - |
acetyl-CoA | mutant E181A, Kcat/KM: 0.9 1/min/mM | Homo sapiens | |
additional information | - |
Mg2+ | mutant E181Q, Kcat/KM: 0.005 1/min/mM | Homo sapiens | |
additional information | - |
acetyl-CoA | mutant E181Q, Kcat/KM: 0.5 1/min/mM | Homo sapiens | |
additional information | - |
Mg2+ | mutant H111A, Kcat/KM: 0.21 1/min/mM | Homo sapiens | |
additional information | - |
acetyl-CoA | mutant H111A, Kcat/KM: 64.2 1/min/mM | Homo sapiens | |
additional information | - |
Mg2+ | mutant K185A, Kcat/KM: 0.016 1/min/mM | Homo sapiens | |
additional information | - |
acetyl-CoA | mutant K185A, Kcat/KM: 0.6 1/min/mM | Homo sapiens | |
additional information | - |
Mg2+ | mutant Q112E, E181Q, Kcat/KM: 0.0002 1/min/mM | Homo sapiens | |
additional information | - |
Mg2+ | mutant Q112E, Kcat/KM: 1.76 1/min/mM | Homo sapiens | |
additional information | - |
acetyl-CoA | mutant Q112E, Kcat/KM: 46.8 1/min/mM | Homo sapiens | |
additional information | - |
Mg2+ | mutant R47A, Kcat/KM: 14.5 1/min/mM | Homo sapiens | |
additional information | - |
acetyl-CoA | mutant R47A, Kcat/KM: 447 1/min/mM | Homo sapiens | |
additional information | - |
acetyl-CoA | mutant R86A, Kcat/KM: 146 1/min/mM | Homo sapiens | |
additional information | - |
Mg2+ | mutant R86A, Kcat/KM: 4.8 1/min/mM | Homo sapiens | |
additional information | - |
Mg2+ | wild-type, Kcat/KM: 32.7 1/min/mM | Homo sapiens | |
additional information | - |
acetyl-CoA | wild-type, Kcat/KM: 578 1/min/mM | Homo sapiens | |
0.0247 | - |
acetyl-CoA | +/-0.0036 mM, mutant K185A | Homo sapiens | |
0.0249 | - |
acetyl-CoA | +/-0.003 mM, wild-type | Homo sapiens | |
0.0454 | - |
acetyl-CoA | +/-0.0038 mM, mutant E181A | Homo sapiens | |
0.047 | - |
acetyl-CoA | +/-0.023 mM, mutant Q112E | Homo sapiens | |
0.076 | - |
acetyl-CoA | +/-0.0038 mM, mutant R47A | Homo sapiens | |
0.081 | - |
acetyl-CoA | +/-0.0013 mM, mutant E181Q | Homo sapiens | |
0.093 | - |
acetyl-CoA | +/-0.001 mM, double mutant Q112E, E181Q | Homo sapiens | |
0.095 | - |
acetyl-CoA | +/-0.033 mM, mutant H111A | Homo sapiens | |
0.142 | - |
acetyl-CoA | +/-0.035 mM, mutant D129A | Homo sapiens | |
0.399 | - |
acetyl-CoA | +/-0.041 mM, mutant R86A | Homo sapiens | |
0.44 | - |
Mg2+ | +/-0.04 mM, wild-type | Homo sapiens | |
0.75 | - |
Mg2+ | +/-0.24 mM, mutant Q112E | Homo sapiens | |
1.1 | - |
Mg2+ | +/-0.1 mM, mutant K185A | Homo sapiens | |
3.3 | - |
Mg2+ | +/-1 mM, mutant R47A | Homo sapiens | |
4.6 | - |
Mg2+ | +/-0.3 mM, mutant D129A | Homo sapiens | |
7.6 | - |
Mg2+ | +/-4.6 mM, mutant E181Q | Homo sapiens | |
15 | - |
Mg2+ | +/-3 mM, mutant R86A | Homo sapiens | |
18.3 | - |
Mg2+ | +/-0.7 mM, mutant E181A | Homo sapiens | |
34.5 | - |
Mg2+ | +/-5.6 mM, mutant H111A | Homo sapiens | |
104.1 | - |
Mg2+ | +/-51 mM, double mutant Q112E, E181Q | Homo sapiens |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | nonessential for binding of coenzyme A (CoA) | Homo sapiens |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
CoA-[4'-phosphopantetheine] + apo-[acyl-carrier protein] | Homo sapiens | - |
adenosine 3',5'-bisphosphate + holo-[acyl-carrier protein] | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | Q9NRN7 | - |
- |
Purification (Comment) | Organism |
---|---|
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 | Homo sapiens |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
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) | Homo sapiens |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
apo-[acyl-carrier protein] + acetyl-CoA | pH 7, 37°C | Homo sapiens | CoA + acetyl-[acyl-carrier protein] | reaction stop by 10% trichloroacetic acid, limited release of 3,5-ADP by interactions with guanidinium moieties of R74 and R86 | ? | |
CoA-[4'-phosphopantetheine] + apo-[acyl-carrier protein] | - |
Homo sapiens | adenosine 3',5'-bisphosphate + holo-[acyl-carrier protein] | - |
? |
Synonyms | Comment | Organism |
---|---|---|
holo ACP synthase | - |
Homo sapiens |
phosphopantetheinyl transferase | close to the class two phosphopantetheinyl transferase Sfp from Bacillus subtilis | Homo sapiens |
PPT | - |
Homo sapiens |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.000098 | - |
acetyl-CoA | mutant D129A | Homo sapiens | |
0.00025 | - |
acetyl-CoA | mutant K185A | Homo sapiens | |
0.0003 | - |
Mg2+ | mutant K185A | Homo sapiens | |
0.00032 | - |
Mg2+ | double mutant Q112E, E181Q | Homo sapiens | |
0.00038 | - |
Mg2+ | mutant E181A | Homo sapiens | |
0.0004 | - |
acetyl-CoA | double mutant Q112E, E181Q | Homo sapiens | |
0.00065 | - |
Mg2+ | mutant E181Q | Homo sapiens | |
0.0007 | - |
Mg2+ | mutant D129A | Homo sapiens | |
0.00072 | - |
acetyl-CoA | mutant E181A | Homo sapiens | |
0.00073 | - |
acetyl-CoA | mutant E181Q | Homo sapiens | |
0.022 | - |
Mg2+ | mutant Q112E | Homo sapiens | |
0.037 | - |
acetyl-CoA | mutant Q112E | Homo sapiens | |
0.102 | - |
acetyl-CoA | mutant H111A | Homo sapiens | |
0.122 | - |
Mg2+ | mutant H111A | Homo sapiens | |
0.24 | - |
Mg2+ | wild-type | Homo sapiens | |
0.24 | - |
acetyl-CoA | wild-type | Homo sapiens | |
0.57 | - |
acetyl-CoA | mutant R47A | Homo sapiens | |
0.8 | - |
Mg2+ | mutant R47A | Homo sapiens | |
0.973 | - |
acetyl-CoA | mutant R86A | Homo sapiens | |
1.2 | - |
Mg2+ | mutant R86A | Homo sapiens |