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Structure and substrate-transfer mechanism of bimodular polyketide megasynthases

English title Structure and substrate-transfer mechanism of bimodular polyketide megasynthases
Applicant Maier Timm
Number 125357
Funding scheme Project funding (Div. I-III)
Research institution Institut für Molekularbiologie und Biophysik ETH Zürich
Institution of higher education ETH Zurich - ETHZ
Main discipline Molecular Biology
Start/End 01.09.2009 - 31.12.2011
Approved amount 274'004.00
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All Disciplines (2)

Discipline
Molecular Biology
Biophysics

Keywords (5)

Multienzyme complex; Macromolecular assembly; X-ray crystallography; Chemical Biology; Combinatorial Biosynthesis

Lay Summary (English)

Lead
Lay summary
Modular polyketide synthase (PKS) megaenzymes are molecular assembly lines for the production of a wide variety of bioactive compounds of outstanding medical relevance, including key drugs, such as erythromycin A (antibiotic), epothilone (anticancer), lovastatin (cholesterol-lowering) and rapamycin (immunosuppressant). The diversity of products is encoded in the sequence and modular structure of PKS enzyme systems. However, most efforts to tailor PKS systems for the production of novel drug candidates have proven inefficient so far, mainly due to a lack of structural understanding of PKS organization. The aim of this project is to reveal the basis of PKS function, the mechanism of intermodular substrate transfer, using the structure determination of intact PKS modules and bimodules as a key method. The resulting insights shall provide a rational basis for future studies of PKS systems and the design of novel biosynthetic assembly lines for drug candidates.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Biology Lecture Series 10.05.2011 Durham, U.K.


Communication with the public

Communication Title Media Place Year
Talks/events/exhibitions Biozentrum Open Door Day German-speaking Switzerland 10.09.2011

Associated projects

Number Title Start Funding scheme
138262 Multienzymes and the Regulation of Eukaryotic Lipid Metabolism 01.01.2012 Project funding (Div. I-III)

Abstract

Polyketide Synthases: Macromolecular assembly lines for drug productionThe modular polyketide synthase (PKS) megaenzymes are molecular assembly lines for the production of a wide variety of bioactive compounds naturally occurring in numerous microorganims, most prominent in Actinomycetales. Their products are of outstanding medical relevance and include key drugs, such as erythromycin A (antibiotic), epothilone (anticancer), lovastatin (cholesterol-lowering) and rapamycin (immunosuppressant). The diversity of products is encoded in the sequence and structure of PKS enzyme systems. These are build from multiple polypeptides, each carrying several complete sets of domains required for the stepwise two-carbon elongation of precursors organized into discrete modules, through which the growing substrates are shuttled in a directed manner. With their size of up to 2MDa per polypeptide and a large number of active sites PKS are amongst the most complex biological machines. The fact that microorganisms carry the burden to produce these huge enzyme systems immediately demonstrates their biological importance. Because of the inefficiency of their chemical synthesis most polyketide drugs are produced biosynthetically; consequently, modified variants are not available via combinatorial chemistry. However, the modular architecure of PKS offers the opportunity for combinatorial biosynthesis, i.e. for the creation of novel polyketide assembly lines by shuffling and modification of domains or modules. Currently, high-throughput molecular biology techniques are employed in this process to produce novel drug candidates. The success of these attempts is rather limited; to a great extent because of a lack of understanding of PKS architecture. While reaction mechanisms of individual domains are well known, their arrangement into assembly lines remains enigmatic. Currently, no structures are available of full PKS proteins, and not even of isolated intact modules. Structure and substrate transfer mechanism of bimodular polyketide megasynthases Encouraged by our recent success in the structure determination of fatty acid synthases (FAS), I propose to determine the structure of a bimodular PKS by X-ray crystallography. This structure shall reveal the arrangement of domains within PKS modules and the structural principles governing directed intermodular substrate transfer, the key feature of modular PKS. To achieve this I am collaborating with Prof. P. Leadlay, Cambridge,U.K., who provides unique expression strains for PKS production and with the in situ crystallization facility of the Swiss Light Source (SLS), Villigen, Switzerland, for high-throughput crystallization screening with in situ X-ray analysis. Recombinantly expressed PKS protein is already available in sufficient amounts and quality for crystallization and initial crystallization screens were carried out. Further possibilities for the production of additional PKS enzymes have been explored, but in light of the success of recombinant expression they only serve as a backup. As a next step, the screening space for crystallization will be narrowed down and promising lead conditions are further optimized by narrow screening of the mutli-dimensional crystallization space. Mechanism-based conformational stabilization has been established in the lab of Prof. Leadlay and is immediately available to produce sufficient amounts of protein for crystallization. Moreover, targeted disulfide crosslinking can be conduct with the available recombinant material. If none of these approaches would yield diffraction quality crystals, additional constructs and proteins purified from their native source strains are available as further targets. In this case, the current samples would still be employed to obtain low-resolution representations by electron microscopy and small-angle X-ray scattering. In a hybrid approach, our very recent atomic structural model of mammalian FAS is now available to interpret such low resolution data in terms of a pseudo-atomic model, which would still allow relevant conclusions on the modular organization of PKS. Once diffracting crystals are available, crystallographic data are collected at SLS and an atomic or pseudo-atomic structural model of a bimodular PKS will be built and used to describe the substrate transfer in modular PKS. Significance of structural insights into the substrate-transfer mechanisms in PKS The expected results will provide detailed structural insights into the biology of PKS assembly lines and also allow conclusion on other conceptually related megasynthases. Importantly, they will contribute to the biocombinatorial design and production of novel polyketide-based drugs by providing a rational basis for tailoring of PKS assembly lines.
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