The working concept of the fungal fatty acid synthase (FAS)
Although large multifunctional enzyme assemblies are used for some of the most important biochemical pathways found in nature, very little is known about their structure, evolution and mechanism of assembly. The megasynthases are a group of multienzymes that perform iterative condensation reactions. This protein class contains large complexes like the fatty acid synthases (FAS) and the polyketide synthases (PKS) for condensation of carboxylic acids, as well as the nonribosomal peptide synthetases (NRPS) for condensation of amino acids. The highly compartmentalized architecture of these multienzymes increases the rate of synthesis by substrate channeling, achieving high local concentrations of intermediates.
We have recently determined the structure of the Saccharomyces cerevisiae FAS type I multienzyme using X-ray crystallography assisted by cryo electron microscopy reconstructions, to a resolution of 4 Å. The S. cerevisiae fatty acid represents the first large multidomain protein solved to atomic resolution (Jenni et al., (2007) Science; Lomakin et al., (2007) Cell; P. Johansson et al., (2008) PNAS).
Figure. Different systems of fatty acid synthesis.
Nature uses two different strategies for the synthesis of fatty acids. Most bacteria synthesize fatty acids by a set of discrete enzymes, each catalyzing one step of the fatty acid synthesis cycle (FAS type II, right figure). In contrast, eukaryotes and some bacteria use large multifunctional enzymes that catalyze all necessary steps (FAS type I). Representing the class of multifunctional FAS I complexes, the mammalian (middle figure) and the fungal fatty acid synthase (right figure) are abstracted.
Properties of fatty acids and fatty acids synthesis
The polar carboxyl group and the unpolar aliphatic tail dictate the amphipathic character of fatty acids.
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The structure of the Saccharomyces cerevisiae FAS
The fungal FAS is a large 2.6 MDa multienzyme complex built from two different polyfunctional proteins, α and β, harboring all the functional domains necessary for the synthesis of long acyl chains.
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The ketoacyl synthase is the heart of the FAS
The condensation step, performed by the ketoacyl synthase (KS), is the key step in the fatty acid synthesis cycle. Subsequent reduction and dehydration reactions prepare the growing chain for the next condensation step.
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Inhibition of the FAS multienzyme
The central role of fatty acids in living entities makes fatty acid synthesis a relevant target for antineoplastic and antibiotic drugs.
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Activation of the Saccharomyces cerevisiae FAS
In crystallographic structures of the S. cerevisiae FAS all functional domains could be identified as directing their active sites towards the inner volume of the barrel. The ACP domain is flexibly attached in the inner compartment and delivers substrates and intermediates to the catalytic centers. Intriguingly, the PPT domain could be determined to be positioned on the exterior surface of the FAS barrel, separated by 60 Å from the ACPs in the reaction chambers.
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Structure and function of the flavoprotein dodecin
Dodecin is a flavin binding protein which was first found in the archaeal organism
Halobacterium salinarum. It forms a dodecameric, hollow-spherical particle of a diameter of about 7 nm
. A single protein consists of 68 amino acids, representing the smallest flavoprotein known to date. Sequence alignments reveal that the dodecin gene is widely distributed within bacteria and haloarchaea. Interestingly, a significant number of bacteria bearing dodecins are human pathogens and exposed to an increased oxygen concentration or radical stress through host defense.
Figure. Reaction routes of flavins.
Flavins are highly reactive compounds. In their unbound state, they can undergo a wealth of reactions, abstracted by bold arrows. The reaction pathways are determined by the surrounding environment, e.g. redox potential, light, reaction partner, and generally proceed in an unspecific manner (left figure). Flavoenzymes bind flavins and select for one reaction route by modulating the flavin chemistry. The flavoenzyme is abstracted by a yellow vessel. A single reaction proceeds (middle figure). Riboflavin binding proteins (RfBPs) are different to flavoenzymes. They store riboflavin, and in order to not harm the cell by a pool of these reactive compounds, they suppress flavin reactivity (right figure).
Properties of flavins
Flavins transform physical input into biological output signals. This ability is based on the broad chemistry of flavins.
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The structure of dodecin
Dodecin exhibits a new fold of a simple β1-α-β2-β3-topology, comprising an α-helix which is partly enwrapped by a three stranded antiparallel β-sheet.
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Binding of FAD to H. salinarum dodecin
FAD was found to saturate dodecin binding pockets at a ligand/binding position ratio of 0.5.
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The function of dodecin in H. salinarum
Prosthetic groups are compounds which are covalently or with high affinity bound to protein. These compounds enhance the proteins’ (bio)chemical repertoire to turn physical input into biological output signals. However, there is one major class of flavin binding proteins which contradicts this concept. Riboflavin binding proteins (RfBPs) have established in nature to uncouple the mediation of signals.
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Biotechnological applications of dodecin
The knowledge about the dodecin functionality was continuously fed into the project of switching dodecin between a holo- and a apoprotein state on electrochemically active surfaces. The sensitivity of dodecin towards the redox-state of the bound flavin represents one of the key characteristics in this approach. While dodecin binds oxidized flavins with high affinities, reduced compounds can not be adopted. The switching of protein states on surfaces requires a tethered flavin (flavin/molecular wire hybrid) which enables the electron transfer from the surface (electrode) to the flavin head group (isoalloxazine ring)
We envision using dodecin as a molecular digger in a single molecule approach. A single flavin covalently connected to the conductive tip of an atomic force microscope, and electric contacting of the cantilever, potentially enables to grab (oxidized flavin), position (oxidized flavin) and release (reduced flavin) individual dodecin molecules.
Figure. Electrochemically switching of protein states.
Flavin/molecular wire ligand hybrids are linked to electrode surfaces. The flavin group is switched between an oxidized (left; flavin abstracted as yellow ellipse, molecular wire in red) and a reduced state (right, yellow rectangle) via a electric potential which is applied onto an electrochemically active surface (electrodes abstracted as black circles). The sensitivity of dodecin towards the redox-state of the bound flavin allows to trigger a surface-bound holoprotein and a free apoprotein state.
Synthesis of electrochemically active surfaces
In the approach of switching dodecin on electrode surfaces, the crucial component is the flavin/molecular wire hybrid ligand (research performed by Gilbert Nöll, University Siegen).
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Protein engineering to optimize dodecin characteristics
Complementary to the flavin/molecular wire hybrid ligand, dodecin properties are optimized by protein engineering.
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Dodecin for blue-light-triggered photorelease
Switching the flavin between an oxidized and a reduced state can be used to trigger the change of dodecin between a loaded and an unloaded state. Flavin reduction can be induced by irradiation with blue light in presence of EDTA.
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