Reaction mechanism of iterative minimal polyketide synthases (PKS) Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life.

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)


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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


Domains in a minimal polyketide syntase

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CoA S

Coenzym A (CoA)


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)


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SAT

ACP

KS

TE

SH

SH

SH

SH

CoA SLoading of a starter unit C

O

CH3


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S

CoA SH

C

O

CH3AT

ACP

KS

TE

S

SH

SH

Loading of a starter unit

SH


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S

SH

CoA SH

C

O

CH3

AT

ACP

KS

TE

SSH

SH


SH


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SH

CoA SH

C

O

CH3

AT

ACP

KS

TE

SH

S

SH


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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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


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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)


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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


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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


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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


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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


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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


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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


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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


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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


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


SH


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S

KS

TE

S

CoA SH

SHAT

ACP S

C

O

CH2

C

O

CH3

C

O

CH2

SH


SH


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S

KS

TE

S

CoA SH

SHAT

ACP S

SH


SH

C

O

CH2

C

O

CH3

C

O

CH2


Next

S

KS

TE

S

CoA SH

SHAT

ACP S

SH


SH

SH

C

O

CH2

C

O

CH3

C

O

CH2

HO


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S

KS

TE

S

CoA SH

SHAT

ACP S

SH


SH

SH

C

O

CH2

C

O

CH3

C

O

CH2Starter unit

1st extender unit

2nd extender unit

Rasmus J.N. Frandsen 2007

HO


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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


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?


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

Reaction mechanism of iterative minimal polyketide synthases (PKS) Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life.

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