Structural Basis of the Substrate-specific Two-step Catalysis of Long Chain Fatty Acyl-CoA Synthetase Dimer*

Long chain fatty acyl-CoA synthetases are responsible for fatty acid degradation as well as physiological regulation of cellular functions via the production of long chain fatty acyl-CoA esters. We report the first crystal structures of long chain fatty acyl-CoA synthetase homodimer (LC-FACS) from Thermus thermophilus HB8 (ttLC-FACS), including complexes with the ATP analogue adenosine 5′-(β,γ-imido) triphosphate (AMP-PNP) and myristoyl-AMP. ttLC-FACS is a member of the adenylate forming enzyme superfamily that catalyzes the ATP-dependent acylation of fatty acid in a two-step reaction. The first reaction step was shown to propagate in AMP-PNP complex crystals soaked with myristate solution. Myristoyl-AMP was identified as the intermediate. The AMP-PNP and the myristoyl-AMP complex structures show an identical closed conformation of the small C-terminal domains, whereas the uncomplexed form shows a variety of open conformations. Upon ATP binding, the fatty acid-binding tunnel gated by an aromatic residue opens to the ATP-binding site. The gated fatty acid-binding tunnel appears only to allow one-way movement of the fatty acid during overall catalysis. The protein incorporates a hydrophobic branch from the fatty acid-binding tunnel that is responsible for substrate specificity. Based on these high resolution crystal structures, we propose a unidirectional Bi Uni Uni Bi Ping-Pong mechanism for the two-step acylation by ttLC-FACS.

[1]  P. Brick,et al.  Structural basis for the activation of phenylalanine in the non‐ribosomal biosynthesis of gramicidin S , 1997, The EMBO journal.

[2]  Liang Tong,et al.  Crystal structure of yeast acetyl-coenzyme A synthetase in complex with AMP. , 2004, Biochemistry.

[3]  A. Guranowski,et al.  Adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate are synthesized by yeast acetyl coenzyme A synthetase , 1994, Journal of bacteriology.

[4]  Makoto Ojika,et al.  Firefly luciferase is a bifunctional enzyme: ATP‐dependent monooxygenase and a long chain fatty acyl‐CoA synthetase , 2003, FEBS letters.

[5]  Paul N. Black,et al.  Transmembrane Movement of Exogenous Long-Chain Fatty Acids: Proteins, Enzymes, and Vectorial Esterification , 2003, Microbiology and Molecular Biology Reviews.

[6]  J. Bar-Tana,et al.  Palmitoyl-coenzyme A synthetase. Mechanism of reaction. , 1973, The Biochemical journal.

[7]  J. Rothman,et al.  Fatty acylation promotes fusion of transport vesicles with Golgi cisternae , 1990, The Journal of cell biology.

[8]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[9]  Ida Schomburg,et al.  Springer handbook of enzymes , 2006 .

[10]  J. Shockey,et al.  Two long-chain acyl-CoA synthetases from Arabidopsis thaliana involved in peroxisomal fatty acid beta-oxidation. , 2002, The Plant journal : for cell and molecular biology.

[11]  C. Saunders,et al.  Evidence for a single non-arachidonic acid-specific fatty acyl-CoA synthetase in heart which is regulated by Mg2+. , 1996, The Biochemical journal.

[12]  T. N. Bhat,et al.  The Protein Data Bank , 2000, Nucleic Acids Res..

[13]  A. Horswill,et al.  The 1.75 A crystal structure of acetyl-CoA synthetase bound to adenosine-5'-propylphosphate and coenzyme A. , 2003, Biochemistry.

[14]  J. Rothman,et al.  Possible role for fatty acyl-coenzyme A in intracellular protein transport , 1987, Nature.

[15]  A. Vagin,et al.  MOLREP: an Automated Program for Molecular Replacement , 1997 .

[16]  P. Brick,et al.  Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. , 1996, Structure.

[17]  D. V. van Aalten,et al.  The structural basis of acyl coenzyme A‐dependent regulation of the transcription factor FadR , 2001, The EMBO journal.

[18]  K. Sugano.  Susceptibility of ganglioside GM1 to a new bacterial neuraminidase , 1978, FEBS letters.

[19]  D. Higgins,et al.  T-Coffee: A novel method for fast and accurate multiple sequence alignment. , 2000, Journal of molecular biology.

[20]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[21]  P. Black,et al.  Vectorial Acylation in Saccharomyces cerevisiae , 2003, The Journal of Biological Chemistry.

[22]  P. Lazarow,et al.  Acyl-CoA synthetase in rat liver peroxisomes. Computer-assisted analysis of cell fractionation experiments. , 1980, The Journal of biological chemistry.

[23]  Thomas C. Terwilliger,et al.  Electronic Reprint Biological Crystallography Maximum-likelihood Density Modification , 2022 .

[24]  David Eisenberg,et al.  3D domain swapping: As domains continue to swap , 2002, Protein science : a publication of the Protein Society.

[25]  P. Berg.  Acyl adenylates; an enzymatic mechanism of acetate activation. , 1956, The Journal of biological chemistry.

[26]  J. Bar-Tana,et al.  The purification and properties of microsomal palmitoyl-coenzyme A synthetase. , 1971, The Biochemical journal.

[27]  E. Yamashita,et al.  Trichromatic concept optimizes MAD experiments in synchrotron X-ray crystallography. , 2002, Structure.

[28]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[29]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[30]  P. R. Sibbald,et al.  The P-loop--a common motif in ATP- and GTP-binding proteins. , 1990, Trends in biochemical sciences.

[31]  M. Osumi,et al.  Subcellular localization of two long-chain acyl-coenzyme-A synthetases in Candida lipolytica. , 1978, European journal of biochemistry.

[32]  J. Berger,et al.  Murine bubblegum orthologue is a microsomal very long-chain acyl-CoA synthetase. , 2004, The Biochemical journal.

[33]  J. Bar-Tana,et al.  Palmitoyl-coenzyme A synthetase. Isolation of an enzyme-bound intermediate. , 1973, The Biochemical journal.

[34]  P. Black,et al.  Functional Role of Fatty Acyl-Coenzyme A Synthetase in the Transmembrane Movement and Activation of Exogenous Long-chain Fatty Acids , 2002, The Journal of Biological Chemistry.

[35]  B. Bahnson,et al.  Five coplanar anion binding sites on one face of phospholipase A2: relationship to interface binding. , 2001, Biochemistry.

[36]  W. Cleland.  The kinetics of enzyme-catalyzed reactions with two or more substrates or products. I. Nomenclature and rate equations. , 1963, Biochimica et biophysica acta.

[37]  Thomas C. Terwilliger,et al.  Automated MAD and MIR structure solution , 1999, Acta crystallographica. Section D, Biological crystallography.

[38]  P. Watkins,et al.  Disruption of the Saccharomyces cerevisiae FAT1 Gene Decreases Very Long-chain Fatty Acyl-CoA Synthetase Activity and Elevates Intracellular Very Long-chain Fatty Acid Concentrations* , 1998, The Journal of Biological Chemistry.

[39]  M. Marahiel,et al.  Crystal structure of DhbE, an archetype for aryl acid activating domains of modular nonribosomal peptide synthetases , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[40]  J. Lai,et al.  Differential effects of fatty acyl coenzyme A derivatives on citrate synthase and glutamate dehydrogenase. , 1993, Research communications in chemical pathology and pharmacology.

[41]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[42]  K. Sugawara,et al.  The sites for fatty acylation, phosphorylation and intermolecular disulphide bond formation of influenza C virus CM2 protein. , 2001, The Journal of general virology.

[43]  Rajesh S. Gokhale,et al.  Enzymic activation and transfer of fatty acids as acyl-adenylates in mycobacteria , 2004, Nature.

[44]  P. Black,et al.  Affinity Labeling Fatty Acyl-CoA Synthetase with 9-p-Azidophenoxy Nonanoic Acid and the Identification of the Fatty Acid-binding Site* , 2000, The Journal of Biological Chemistry.

[45]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .

[46]  T. Tanaka,et al.  Acyl-coenzyme-A synthetase I from Candida lipolytica. Purification, properties and immunochemical studies. , 1979, European journal of biochemistry.

[47]  Antje Chang,et al.  New Developments , 2003 .

[48]  Richard J Morris,et al.  ARP/wARP's model-building algorithms. I. The main chain. , 2002, Acta crystallographica. Section D, Biological crystallography.

[49]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[50]  Yutaka Kuroda,et al.  Structural genomics projects in Japan , 2000, Nature Structural Biology.

[51]  W. O'brien,et al.  Evidence for a complex of three beta-oxidation enzymes in Escherichia coli: induction and localization , 1977, Journal of bacteriology.

[52]  Maria Jesus Martin,et al.  The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003 , 2003, Nucleic Acids Res..