architectures of mammalian and fungal fatty acid synthases presentation based on: t. maier, s....

Architectures of Mammalian and Fungal Fatty Acid Synthases

Presentation based on:

T. Maier, S. Jenni, N. Ban, Science 311, 1258 (2006). -- Mammalian fatty acid at 4.5 Å resolution

S. Jenni, M. Leibundgut, T. Maier, N. Ban, Science 311, 1263 (2006). -- Fungal fatty acid at 5 Å resolution

Agenda

1. fatty acid quick peak

2. catalytic cycle of fatty acid synthesis

3. mammalian fatty acid synthase structure

4. fungal fatty acid synthase structure

QuickTime™ and aTIFF (Uncompressed) decompressor

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1. Fatty acid quick peak

Common fatty acids are carboxylic acids with long hydrocarbon tails:

comes with a COOH head and a tail of many CH2.

2. Fatty acid catalytic cycle

2.1 Common elongation scheme

starter substrates Acetyl coenzyme A (Acetyl-CoA) and Malonyl-CoA transfer the active functionals to acyl carrierprotein (ACP).

ACP transports substrate to different reaction sites, catalyzed by different enzyme. A complete cycle gives the acyl group an additional two carbon units.

This step-wise elongation repeats until a substrate length of C16 to C18 is achieved.

Another enzyme then release the substrate from ACP, completing the synthesis process.

2.2 Step-by-step details of catalytic cycle



: catalysts for different individual reactions

protein that releases the completed product from ACP.

A

B

C

DE

F

2.3 Different fatty acid synthase (FAS) systems

Type II FAS (bacteria) -- all reactions carried out by individual, monofunctional proteins.

Type I FAS (eukaryote) -- large, multifuctional polypeptides contains all necessary enzymes for the enlongation cycle.

Fungal FAS: 2.6-MD 66 dodecamer, catalytic domains distributed over two distinct subunits.

Vertebrates and Mammal FAS: 270-kD 2 homodimer, contains all catalytic activities.

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3. Mammalian fatty acid synthase

3.1 Functional proteins



ACP: acyl protein carrier

MAT: malonyl-CoA-/acetyl- CoA-ACP-transacylase

KS: -ketoacyl synthase

KR: -ketoacyl reductase

DH: dehydratase

ER: -enoyl reductase

TE: thioesterase

3.2 overall structure and domain assignment



210Å 180Å 90Å

Blue bubble:Electron density cloud viaX-ray crystallography

Colored proteins:Identified as specific domainvia mapping homologous protein structures

3.2.1 KS domain



Mammalian KS closely resembles the Escherichia coli KS I (FabB).So KS domain was fitted with E. coli FabB.





3.2.2 MAT domain

Mammalian MAT is homologous to bacterial malonyl transferase (FabD).So we fit the MAT domain with Streptomyces coelicolor FabD.





3.2.3 DH domain

Mammalian DH adopts a “double hot dog” fold that’s closely related to the fold of the dimeric bacterial dehydratases FabA and FabZ. So we fit DH with two monomers of dimeric E. coli FabA.





3.2.4 ER domain

The best structural match for ER was obtained with a zinc-free bacterial quinone reductase. Here the particular model is quinone reductase of T. thermophilus.





3.2.5 KR domain

KR belongs to the short-chain dehydrogenase family, and was modeled with E. coli FabG.



3.2.6 ACP and TE domain

* ACP and TE domains could not be placed with confidence, likely due to their inherent flexibility.

* However…



This blurred volume of electron density, which was observed only onone side, might be interpreted as arising from the C-terminal ACPand TE domains.



3.2.7 Table of structural and functional analogs

3.3 intersubunit and interdomain connections

FAS is an intertwined dimer with a large dimerization interface.


are needed to see this picture.KS domain dimerize in the same way as homologous homodimeric FabB.




are needed to see this picture.The interaction betweenER monomers are guidedby the formation of a continuous 12-stranded-sheet. The same way as the homologousbacterial enzyme.





There are other substantialintersubunit contacts in the unassigned region of electrondensity map.

COLORED: identified domains

GREY: unassigned region.

3.4 active sites and the two reaction chambers


are needed to see this picture.QuickTime™ and a

TIFF (Uncompressed) decompressorare needed to see this picture.

Solid spheres: active sites

Hollow spheres: radii = length of the phosphopantheteine arm of ACP

4. Fungal fatty acid synthase

4.1 Functional proteins

ACP: acyl protein carrier

MPT: malonyl/palmitoyl transferase

KS: ketoacyl synthase

KR: ketoacyl reductase

DH: dehydratase

ER: enoyl reductase

AT: acetyl transferase

PT: phosphopantetheine transferase (for ACP activation)



4.2 overall structure and domain assignment 230Å 230Å 260Å



MPT: malonyl/palmitoyl transferase

KS: ketoacyl synthase

KR: ketoacyl reductase

DH: dehydratase

ER: enoyl reductase

AT: acetyl transferase

ACP and PT structures could not be identified.

White regions denote unidentified electron density.

4.2.1 KS domain





KS dimer domain was identified by finding the thiolase fold in the FAS electron density map (bacterial KS is known to adopt a thiolase fold and form homodimers).

Bacterial KS homolog fits almost perfectly into the density map.



4.2.2 KR domain







The 4-helix bundle is a characteristic trait of one of the dimerization interface in type-II tetrameric KR homolog of Brassica napus.It also contains a Rossmann fold.

The Brassica napus KR homolog fits FASelectron density remarkably well.

4.2.3 DH domain

The closest sequence homolog with known structure of fungal DH is the human Peroxisomal 2-enoyl-CoA hydratase 2 involved in oxidation of fatty acids. It is a pseudo-dimer, has two “hot dog”folds, and forms a large sheet.DH structures of bacterial FAS fits less well, since they are true homodimers.







4.2.4 ER domain

The fungal ER, unlike other FAS systems,is a FMN-containing oxidoreductase, andno homology has been observed.Since a TIM-barrel fold was discovered nearthe FMN-binding pocket, the 21 known TIM-barrel superfamilies were examined, and a good fit was obtained with spinach glycolate oxidase.







4.2.5 AT and MPT



AT

MPT





AT and MPT are homologous in sequence, catalyze similar reactions, and have same protein fold. Therefore they are both fittedwith malonyl transferase from Streptomycescoelicolor.



In order to unambiguously assign AT andMPT domain, the locations of their N termini relative to the C terminus of DHwere observed.





4.3 The reaction chambers





** Two identical reaction chambers separated by central wheel.

** Each contains three copies of a full set of catalytic domains.

** All active sites are oriented towards interior. Red cones indicate entrances to active sites.

COLORED: fitted domainsGREY: unassigned region.






A set of active sites in the reaction chamber with allenzymatic activities required for the synthesis cycle.

Green sphere: reaction chamber center.




Schematic path of ACP, shuttlingsubstrate between the active sites.





architectures of mammalian and fungal fatty acid synthases presentation based on: t. maier, s....

Documents

resolution slide

fatty acid catalytic

thioesterase slide

ks domain mammalian

er domain

mat domain mammalian

dh domain mammalian

specific domain