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Reaction mechanism of iterative minimal polyketide synthases (PKS) PowerPoint PPT Presentation


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Reaction mechanism of iterative minimal polyketide synthases (PKS).

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Reaction mechanism of iterative minimal polyketide synthases (PKS)

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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


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Domains in a minimal polyketide syntase

AT

AT domain = Acyltransferase

SH

ACP

SH

Acyl Carrier protein (ACP)

KS

SH

b-ketoacyl synthase (KS)

TE

SH

Thioesterase (TE)

Next

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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Domains in a minimal polyketide syntase

AT

AT domain = Acyltransferase

SH

Prosthetic group: 4-phosphopantetheine (PPT). A flexible group that can transfer the starter and extender units internally in the enzyme.

ACP

SH

Acyl Carrier protein (ACP)

KS

SH

b-ketoacyl synthase (KS)

TE

SH

Thioesterase (TE)

Next

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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CoA

S

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.

Adenin

Ribo-3’-phosphat

4-phosphopantetheine

=

Next

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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CoA

S

Loading of a starter unit

Starter unit

(acetyl-CoA)

AT

SH

ACP

SH

KS

SH

TE

SH

Next

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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CoA

S

Loading of a starter unit

AT

S

SH

ACP

SH

KS

SH

TE

SH

Next

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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Loading of a starter unit

CoA

SH

AT

SH

S

S

ACP

KS

SH

TE

SH

Next

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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Loading of a starter unit

CoA

SH

AT

SH

ACP

SH

S

KS

S

SH

TE

SH

Next

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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Loading of a starter unit

CoA

SH

AT

SH

ACP

SH

KS

S

TE

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.

Next

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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CoA

CoA

S

S

+ CO2

Activation of extender units

Acetyl-CoA

Acetyl-CoA Carboxylase

The CO2 originates from a HCO3- bondto biotin in the enzyme

Malonyl-CoA

Next

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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Extender unit

(malonyl-CoA)

Loading of a extender unit

CoA

S

AT

SH

ACP

SH

KS

S

TE

SH

Next

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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Loading of a extender unit

CoA

S

SH

AT

S

SH

ACP

SH

KS

S

TE

SH

Next

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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Loading of a extender unit

CoA

SH

AT

SH

S

ACP

S

SH

KS

S

TE

SH

Next

Rasmus J.N. Frandsen 2007

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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Ready for condensation

CoA

SH

Decarboxylation of the extender unit (malonyl) provides the energy/electron for the condensation

AT

SH

ACP

S

KS

S

TE

SH

Next

Rasmus J.N. Frandsen 2007

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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Condensation

CoA

SH

Decarboxylation of the extender unit (malonyl) provides the energy/electorne for the codensation

AT

SH

ACP

S

2

KS

S

SH

TE

SH

Next

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences

Rasmus J.N. Frandsen 2007


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Preparing for a second round

CoA

SH

AT

SH

ACP

S

SH

KS

SH

S

TE

SH

Next

Rasmus J.N. Frandsen 2007

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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Loading of the 2nd extender unit

CoA

S

SH

AT

S

SH

ACP

S

SH

KS

S

TE

SH

Next

Rasmus J.N. Frandsen 2007

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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Loading of the 2nd extender unit

CoA

SH

AT

SH

S

ACP

S

SH

KS

S

TE

SH

Next

Rasmus J.N. Frandsen 2007

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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2nd condensation

CoA

SH

Decarboxylation

AT

SH

ACP

S

KS

S

SH

TE

SH

Next

Rasmus J.N. Frandsen 2007

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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Release from the enzyme

CoA

SH

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.

AT

SH

ACP

S

S

KS

S

SH

TE

S

SH

Next

Rasmus J.N. Frandsen 2007

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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Release from the enzyme

CoA

SH

AT

SH

ACP

SH

S

KS

S

SH

TE

S

SH

Next

Rasmus J.N. Frandsen 2007

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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Release from the enzyme

CoA

SH

AT

SH

ACP

S

SH

KS

S

SH

TE

S

Next

Rasmus J.N. Frandsen 2007

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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Release from the enzyme

CoA

SH

AT

SH

ACP

S

SH

KS

S

SH

TE

S

Next

Rasmus J.N. Frandsen 2007

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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Release from the enzyme

CoA

SH

AT

SH

ACP

S

SH

KS

S

SH

TE

SH

S

HO

Next

Rasmus J.N. Frandsen 2007

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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Starter unit

2nd extender unit

1st extender unit

Release from the enzyme

CoA

SH

AT

SH

ACP

SH

S

KS

SH

S

TE

S

SH

HO

Next

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences

Rasmus J.N. Frandsen 2007


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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.

HO

Starter unit

1st extender unit

2nd extender unit

Next

Rasmus J.N. Frandsen 2007

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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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:

b-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

END

Rasmus J.N. Frandsen 2007

Rasmus J.N. Frandsen 2007 ([email protected]) University of Copenhagen, Faculty of Life Sciences


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