Animation of a minimal type I Iterative PKS in action

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Reaction mechanism of
iterative minimal polyketide synthases (PKS)
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.
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Domains in a minimal polyketide syntase
AT domain = Acyltransferase
AT
SH
SH
Acyl Carrier protein (ACP)
ACP
b-ketoacyl synthase (KS)
KS
SH
Thioesterase (TE)
TE
SH
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Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Domains in a minimal polyketide syntase
AT domain = Acyltransferase
AT
SH
SH
Acyl Carrier protein (ACP)
ACP
b-ketoacyl synthase (KS)
KS
SH
Thioesterase (TE)
TE
SH
O
S er
H2
C
Prosthetic group:
4-phosphopantetheine
(PPT). A flexible group that
can transfer the starter and
extender units internally in
the enzyme.
O
P
H 3C
CH3
O
O
SH
N
H
OH
ACP
O
N
H
OH
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Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Coenzym A (CoA)
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.
NH2
N
N
O
N
N
H 2C
O
O
P
O
P
H 3C
CH3
O
O
SH
O
OH
O
N
H
OH
N
H
OH
OH
O
O
P
OH
OH
Adenin
Ribo-3’-phosphat
4-phosphopantetheine
=
CoA
S
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Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
O
Loading of a starter unit
CoA
AT
S
C
CH3
Starter unit
(acetyl-CoA)
SH
SH
ACP
KS
SH
TE
SH
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Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
O
Loading of a starter unit
CoA
AT
S
C
CH3
S
SH
SH
ACP
KS
SH
TE
SH
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Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Loading of a starter unit
CoA
SH
O
AT
SH
S
C
S
CH3
ACP
KS
SH
TE
SH
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Loading of a starter unit
CoA
AT
SH
SH
ACP
O
SH
S
KS
S
SH
TE
SH
C
CH3
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Loading of a starter unit
CoA
AT
SH
SH
ACP
SH
O
KS
S
TE
SH
C
CH3
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.
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Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Activation of extender units
O
Acetyl-CoA
CoA
S
C
CH3
The CO2 originates from a
HCO3- bond to biotin in the
enzyme
Malonyl-CoA
+
CO2
Acetyl-CoA Carboxylase
CoA
S
O
O
C
C
OH
C
H2
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Loading of a extender unit
CoA
S
O
O
C
C
OH
C
H2
AT
Extender unit
(malonyl-CoA)
SH
ACP
SH
O
KS
S
TE
SH
C
CH3
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Loading of a extender unit
CoA
S
SH
O
O
C
C
OH
C
H2
AT
S
SH
ACP
SH
O
KS
S
TE
SH
C
CH3
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Loading of a extender unit
CoA
AT
SH
S
SH
O
O
C
C
OH
C
H2
S
SH
ACP
O
KS
S
TE
SH
C
CH3
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Ready for condensation
Decarboxylation of the extender
unit (malonyl) provides the
energy/electron for the
condensation
CoA
AT
SH
SH
ACP
S
O
O
C
C
C
H2
O
KS
S
TE
SH
C
O-
CH3
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Condensation
Decarboxylation of the extender
unit (malonyl) provides the
energy/electorne for the
codensation
CoA
AT
SH
SH
ACP
S
O
OO
C
CC
C
H2
O
KS
S
SH
TE
SH
C
OO-
CH3
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Preparing for a second round
CoA
AT
SH
SH
O
ACP
S
SH
O
C
C
H2
KS
SH
S
TE
SH
C
CH3
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Loading of the 2nd extender unit
CoA
S
SH
O
O
C
C
OH
C
H2
AT
SH
ACP
S
SH
O
KS
S
O
C
C
H2
TE
C
CH3
SH
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Loading of the 2nd extender unit
CoA
AT
SH
S
SH
O
O
C
C
OH
C
H2
S
SH
ACP
O
KS
S
O
C
C
H2
TE
C
CH3
SH
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
2nd condensation
CoA
SH
Decarboxylation
AT
SH
ACP
S
O
O
C
C
CH2
O
KS
S
SH
O
C
C
H2
TE
O
C
CH3
SH
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Release from the enzyme
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.
CoA
AT
SH
SH
O
ACP
S
O
C
CH2
KS
S
SH
TE
S
SH
O
C
C
H2
C
CH3
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Release from the enzyme
CoA
AT
SH
SH
O
ACP
S
SH
O
C
CH2
KS
S
SH
TE
S
SH
O
C
C
H2
C
CH3
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Release from the enzyme
CoA
AT
SH
SH
ACP
KS
S
SH
S
SH
O
TE
S
O
C
CH2
O
C
C
H2
C
CH3
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Release from the enzyme
CoA
AT
SH
SH
ACP
KS
S
SH
S
SH
O
TE
S
O
C
CH2
O
C
C
H2
C
CH3
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Release from the enzyme
CoA
AT
SH
SH
ACP
KS
S
SH
S
SH
O
TE
S
SH
O
C
CH2
HO
O
C
C
H2
C
CH3
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Release from the enzyme
CoA
AT
SH
SH
ACP
S
SH
KS
S
SH
TE
S
SH
O
HO
2nd
O
C
extender unit
CH2
1st extender
unit
O
C
C
H2
C
Starter unit
CH3
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Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Release from the enzyme
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.
O
HO
O
C
CH2
C
O
C C
H2
CH3
Next
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
Where does the diversity originate from?
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:
O
b-ketoacyl reductase (KR)
C
OH
CH
H 2C
H 2C
OH
Dehydratase (DH)
CH
CH
HC
H 2C
Enoyl reductase (ER)
CH
Methyltransferase (MET)
CH2
HC
H 2C
OH
O
CH
H 2C
CH3
CH
H 2C
Cyclases (Cyc) – fold the polyketide chain into an aromatic or
macrocyclic compound
+ alternative extender units different from malonyl-CoA
Rasmus J.N. Frandsen 2007 ([email protected])
University of Copenhagen, Faculty of Life Sciences
END
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