Functional and structural characterization of the fatty acid synthase type I from Mycobacterium tuberculosis
Final Report Abstract
In this project, we established the recombinant expression of 2 MDa large bacterial type I FAS proteins in E. coli. This is an important methodological development, which significantly improves the access to this protein for in vitro studies; particularly for putative inhibition studies on the M. tuberculosis FAS. We have further succeeding in solving the cryo-EM structure of M. tuberculosis FAS elucidating structural and conformational properties of the protein. This work allowed to describe bacterial FAS as structurally similar proteins to fungal FAS as expected from sequence alignment that, however, show significant different conformational properties. By working on the related fungal FAS from R. toruloides, we finally gave insight into the evolutionary development of the bacterial/fungal FAS family. X-crystallographic studies turned out to be very challenging. Crystals generally yielded low-resolution datasets, and were additionally affected by pathological twinning disorders. Future efforts will first focus on the improved protein purification, and the cross-linking of samples with specific cross-linkers. Subsequent structural studies will mainly focus on cryo-EM taking advantage of the recent technical developments in the field.
Publications
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(2013) Structure and conformational variability of the Mycobacterium tuberculosis fatty acid synthase multienzyme complex. Structure 21, 1251-1257
Ciccarelli, Luciano; Connell, Sean; Enderle, Mathias; Mills, Deryck J.; Vonck, Janet; Grininger, Martin
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(2014) Perspectives on the evolution, assembly and conformational dynamics of fatty acid synthase type I (FAS I) systems. Curr Opin Struct Biol 25, 49-56
Grininger, Martin
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(2015) Cryo-EM structure of fatty acid synthase (FAS) from Rhodosporidium toruloides provides insights into the evolutionary development of fungal FAS. Protein Sci 24, 987-995
Fischer; Manuel; Rhinow, Daniel; Zhu, Zhiwei; Mills, Deryck J.; Zhao, Zongbao K.; Vonck, Janet; Grininger; Martin
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(2015) Crystallization and X-ray diffraction studies of a complete bacterial fatty-acid synthase type I. Acta Crystallogr F Struct Biol Commun 71,1401-1407
Enderle, Mathias; McCarthy, Andrew; Paithankar, Karthik S.; Grininger, Martin
