reaction mechanism of iterative minimal polyketide synthases (pks) rasmus j.n. frandsen 2007...
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Reaction mechanism of iterative minimal polyketide synthases (PKS)
Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
Polyketide synthases are multidomain enzymes that catalyze the condensation of ketide units (starter unit and extender units) resulting in the formation of polyketides. The reaction is driven by decarboxylation of the extender unit during condensation, which is also known as a Claisen condensation. The motivation for making this animation was that many of our students struggled with understanding how the different substrates and products were moved around inside the PKS, during biosynthesis.
The following slides shows the conceptual reaction mechanism and is not correct in chemical terms with respect to the flow of electrons.
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AT
ACP
KS
TE
SH
SH
SH
SH
Domains in a minimal polyketide syntase
AT domain = Acyltransferase
Acyl Carrier protein (ACP)
-ketoacyl synthase (KS)
Thioesterase (TE)
Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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AT
ACP
KS
TE
SH
SH
SH
SH
AT domain = Acyltransferase
Acyl Carrier protein (ACP)
-ketoacyl synthase (KS)
Thioesterase (TE)
Prosthetic group: 4-phosphopantetheine (PPT). A flexible group that can transfer the starter and extender units internally in the enzyme.
O
OH
O
NH
O
NH
SH
CH3H3C
O P
O
OH
H2CSer
ACP
Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
Domains in a minimal polyketide syntase
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CoA S
Coenzym A (CoA)
Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
O
OH
O
NH
O
NH
SH
CH3H3C
O P
O
OH
O P
O
OH
OH2C
O
P OHO
OH
OH
N
N N
N
NH2
4-phosphopantetheineAdenin Ribo-3’-phosphat
=
Coenzym A also contains a 4-phosphopantetheine group, similar to that found on the ACP domain of PKSs. The terminal thioester group serves at the attachment point for acetyl and malonyl units.
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CoA S C
O
CH3
AT
ACP
KS
TE
SH
SH
SH
SH
Loading of a starter unit Starter unit(acetyl-CoA)
Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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SAT
ACP
KS
TE
SH
SH
SH
SH
CoA SLoading of a starter unit C
O
CH3
Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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S
CoA SH
C
O
CH3AT
ACP
KS
TE
S
SH
SH
Loading of a starter unit
SH
Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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S
SH
CoA SH
C
O
CH3
AT
ACP
KS
TE
SSH
SH
Loading of a starter unit
SH
Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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SH
CoA SH
C
O
CH3
AT
ACP
KS
TE
SH
S
SH
Loading of a starter unit
A starter unit has now been loaded into the KS domain of the PKS and we are ready for loading of the first extender unit.
Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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Activation of extender units
CoA S C
O
CH3 + CO2
Acetyl-CoA Carboxylase
CoA S C
O
CH2
C
O
OH
The CO2 originates from a HCO3- bond to biotin in the enzyme
Acetyl-CoA
Malonyl-CoA
Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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SH
CoA S
C
O
CH3
AT
ACP
KS
TE
SH
S
SH
C
O
CH2
C
O
OHLoading of a extender unit Extender unit(malonyl-CoA)
Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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S
C
O
CH2
C
O
OH
SH
CoA S
C
O
CH3
AT
ACP
KS
TE
SH
S
SH
C
O
CH2
C
O
OHLoading of a extender unit SH
Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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S
S
CoA SH
C
O
CH3
AT
ACP
KS
TE
SH
S
SH
C
O
CH2
C
O
OHSH
Loading of a extender unit
Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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SH
CoA SH
C
O
CH3
AT
ACP
KS
TE
S
S
SH
C
O
CH2
C
O
O-
Ready for condensation
Decarboxylation of the extender unit (malonyl) provides the energy/electron for the condensation
Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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SH
CoA SH
C
O
CH3
AT
ACP
KS
TE
S
S
SH
C
O
O
SH
Decarboxylation of the extender unit (malonyl) provides the energy/electorne for the codensation
Condensation
2
Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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SH
CoA SH
AT
ACP
KS
TE
S
SH
SH
C
O
CH2
C
O
CH3
S
Preparing for a second round
SH
Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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SSHAT
ACP
KS
TE
S
SH
C
O
CH2
C
O
CH3
S
CoA S C
O
CH2
C
O
OHLoading of the 2nd extender unit SH
SH
Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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KS
TE SH
S
CoA SH
S
SAT
ACP SH
C
O
CH2
C
O
OHSH
Loading of the 2nd extender unit
C
O
CH2
C
O
CH3
Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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KS
TE SH
S
CoA SH
SHAT
ACP S C
O
CH2
SH
2nd condensation
C
O
O
C
O
CH2
C
O
CH3
Decarboxylation
Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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S
KS
TE SH
S
CoA SH
SHAT
ACP SC
O
CH2
C
O
CH3
C
O
CH2
SH
Release from the enzyme
S
Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
At this stage the enzyme faces a choice, whether to continue with additional rounds of condensations or to release the polyketide chain from the enzyme.
The number of condensation rounds (iterations) that the individual PKSs perform is at present not predictable. One hypothesis is that the size (volume) of the active site in the KS domain could be the deciding factor for total number of iterations possible.
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S
KS
TE SH
S
CoA SH
SHAT
ACP SC
O
CH2
C
O
CH3
C
O
CH2
SH
Release from the enzyme
SH
Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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S
KS
TE
S
CoA SH
SHAT
ACP S
C
O
CH2
C
O
CH3
C
O
CH2
SH
Release from the enzyme
SH
Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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S
KS
TE
S
CoA SH
SHAT
ACP S
SH
Release from the enzyme
SH
C
O
CH2
C
O
CH3
C
O
CH2
Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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S
KS
TE
S
CoA SH
SHAT
ACP S
SH
Release from the enzyme
SH
SH
C
O
CH2
C
O
CH3
C
O
CH2
HO
Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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S
KS
TE
S
CoA SH
SHAT
ACP S
SH
Release from the enzyme
SH
SH
C
O
CH2
C
O
CH3
C
O
CH2Starter unit
1st extender unit
2nd extender unit
Rasmus J.N. Frandsen 2007
HO
Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
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Release from the enzyme
Starter unit
1 st extender unit
2 nd extender unit
Rasmus J.N. Frandsen 2007
C
O
CH2
C
O
CH3
C
O
CH2
HO
Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
Note that the formed polyketide chain has polarity. With a methyl (-CH3) group at the ”oldest” end and a carboxyl (-COOH) group at the ”newest” end.
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Where does the diversity originate from?
Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences
In addition to the four catalytic domains (AT, ACP, KS and TE) used by the minimal PKS. Other domains can also participate in the biosynthesis:
-ketoacyl reductase (KR)
Dehydratase (DH)
Enoyl reductase (ER)
Methyltransferase (MET)
Cyclases (Cyc) – fold the polyketide chain into an aromatic or macrocyclic compound
+ alternative extender units different from malonyl-CoA
C
H2C
O
CH
H2C
OH
CH
H2C
OHCH
HC
CH
HC
CH2
H2C
CH
H2C
OH
CH
H2C
O CH3
END