Literature summary for 2.7.8.7 extracted from

Bunkoczi, G.; Pasta, S.; Joshi, A.; Wu, X.; Kavanagh, K.L.; Smith, S.; Oppermann, U.
Mechanism and substrate recognition of human holo ACP synthase (2007), Chem. Biol., 14, 1243-1253.

Application

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(Commentary)

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 (Commentary)

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

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

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 [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
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

Localization	Comment	Organism	GeneOntology No.	Textmining

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	nonessential for binding of coenzyme A (CoA)	Homo sapiens

Natural Substrates/ Products (Substrates)

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

Organism	UniProt	Comment	Textmining
Homo sapiens	Q9NRN7	-	-

Purification (Commentary)

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

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	a

Substrates and Products (Substrate)

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

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 [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
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