Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomicroscopy

Yeast fatty acid synthase (FAS) is a 2.6-MDa barrel-shaped multienzyme complex, which carries out cyclic synthesis of fatty acids. By electron cryomicroscopy of single particles we obtained a three-dimensional map of yeast FAS at 5.9-Å resolution. Compared to the crystal structures of fungal FAS, the EM map reveals major differences and new features that indicate a considerably different arrangement of the complex in solution compared to the crystal structures, as well as a high degree of variance inside the barrel. Distinct density regions in the reaction chambers next to each of the catalytic domains fitted the substrate-binding acyl carrier protein (ACP) domain. In each case, this resulted in the expected distance of ∼18 Å from the ACP substrate-binding site to the active site of the catalytic domains. The multiple, partially occupied positions of the ACP within the reaction chamber provide direct structural insight into the substrate-shuttling mechanism of fatty acid synthesis in this large cellular machine.

[1]  James Z. Chadick,et al.  Structure and molecular organization of mammalian fatty acid synthase , 2005, Nature Structural &Molecular Biology.

[2]  S. Wakil,et al.  Quaternary structure of human fatty acid synthase by electron cryomicroscopy , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[3]  T. Steitz,et al.  The Crystal Structure of Yeast Fatty Acid Synthase, a Cellular Machine with Eight Active Sites Working Together , 2007, Cell.

[4]  J. Frank,et al.  A method of focused classification, based on the bootstrap 3D variance analysis, and its application to EF-G-dependent translocation. , 2006, Journal of structural biology.

[5]  R. Henderson,et al.  Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. , 2003, Journal of molecular biology.

[6]  D. Oesterhelt,et al.  Multimeric options for the auto-activation of the Saccharomyces cerevisiae FAS type I megasynthase. , 2009, Structure.

[7]  S. Wakil,et al.  Fatty acid synthesis and its regulation. , 1983, Annual review of biochemistry.

[8]  Francisco J. Asturias,et al.  Conformational Flexibility of Metazoan Fatty Acid Synthase Enables Catalysis , 2008, Nature Structural &Molecular Biology.

[9]  N. Ban,et al.  Architecture of mammalian fatty acid synthase , 2006 .

[10]  P. Penczek,et al.  Structure-Function Relationships of the Saccharomyces cerevisiae Fatty Acid Synthase , 1996, The Journal of Biological Chemistry.

[11]  Daniel Boehringer,et al.  Structure of Fungal Fatty Acid Synthase and Implications for Iterative Substrate Shuttling , 2007, Science.

[12]  Robert Finking,et al.  Biosynthesis of nonribosomal peptides , 2003 .

[13]  W Chiu,et al.  EMAN: semiautomated software for high-resolution single-particle reconstructions. , 1999, Journal of structural biology.

[14]  S. Wakil,et al.  Structure-function relationships of the yeast fatty acid synthase: negative-stain, cryo-electron microscopy, and image analysis studies of the end views of the structure. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Jie J. Zheng,et al.  The structural biology of type II fatty acid biosynthesis. , 2005, Annual review of biochemistry.

[16]  B. Böttcher,et al.  Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy , 1997, Nature.

[17]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[18]  Timm Maier,et al.  Architecture of Mammalian Fatty Acid Synthase at 4.5 Å Resolution , 2006, Science.

[19]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[20]  M. Sternberg,et al.  Protein structure prediction on the Web: a case study using the Phyre server , 2009, Nature Protocols.

[21]  H. Taguchi,et al.  Cryo-EM structure of the native GroEL-GroES complex from thermus thermophilus encapsulating substrate inside the cavity. , 2009, Structure.

[22]  Andrzej Witkowski,et al.  Structural and functional organization of the animal fatty acid synthase. , 2003, Progress in lipid research.

[23]  G. Getz,et al.  Structural Basis for Substrate Delivery by Acyl Carrier Protein in the Yeast Fatty Acid Synthase , 2022 .

[24]  S. Wakil,et al.  Yeast fatty acid synthase: structure to function relationship. , 1985, Biochemistry.

[25]  M van Heel,et al.  A new generation of the IMAGIC image processing system. , 1996, Journal of structural biology.

[26]  D. Byers,et al.  Acyl carrier protein: structure-function relationships in a conserved multifunctional protein family. , 2007, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[27]  Chao Yang,et al.  Estimation of variance in single-particle reconstruction using the bootstrap technique. , 2006, Journal of structural biology.

[28]  D. Oesterhelt,et al.  Inhibition of the fungal fatty acid synthase type I multienzyme complex , 2008, Proceedings of the National Academy of Sciences.

[29]  Nathan A. Baker,et al.  PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations , 2004, Nucleic Acids Res..

[30]  M. Sturm,et al.  Three-Dimensional Electron Microscopy of Individual Biological Objects Part III. Experimental Results on Yeast Fatty Acid Synthetase , 1976 .