Catalysis, specificity, and ACP docking site of Streptomyces coelicolor malonyl-CoA:ACP transacylase.

Malonyl-CoA:ACP transacylase (MAT), the fabD gene product of Streptomyces coelicolor A3(2), participates in both fatty acid and polyketide synthesis pathways, transferring malonyl groups that are used as extender units in chain growth from malonyl-CoA to pathway-specific acyl carrier proteins (ACPs). Here, the 2.0 A structure reveals an invariant arginine bound to an acetate that mimics the malonyl carboxylate and helps define the extender unit binding site. Catalysis may only occur when the oxyanion hole is formed through substrate binding, preventing hydrolysis of the acyl-enzyme intermediate. Macromolecular docking simulations with actinorhodin ACP suggest that the majority of the ACP docking surface is formed by a helical flap. These results should help to engineer polyketide synthases (PKSs) that produce novel polyketides.

[1]  M. Welch,et al.  Structure of the CheY-binding domain of histidine kinase CheA in complex with CheY , 1998, Nature Structural Biology.

[2]  S. Wakil,et al.  Studies on the mechanism of fatty acid synthesis. XXVI. Purification and properties of malonyl-coenzyme A--acyl carrier protein transacylase of Escherichia coli. , 1971, Archives of biochemistry and biophysics.

[3]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[4]  C R Hutchinson,et al.  Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations. , 2001, Biochemistry.

[5]  M. Nardini,et al.  University of Groningen Crystal structure of Pseudomonas aeruginosa lipase in the open conformation-The prototype for family I.1 of bacterial lipases Nardini, , 2000 .

[6]  D. Hopwood.  Genetic Contributions to Understanding Polyketide Synthases , 1998 .

[7]  Steven E. Brenner,et al.  SCOP database in 2002 , 2002 .

[8]  Todd J. A. Ewing,et al.  Critical evaluation of search algorithms for automated molecular docking and database screening , 1997 .

[9]  P. Leadlay,et al.  Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl‐CoA:acyl carrier protein transacylase domains in modular polyketide synthases , 1995, FEBS letters.

[10]  T. A. Jones,et al.  Databases in protein crystallography. , 1998, Acta crystallographica. Section D, Biological crystallography.

[11]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[12]  C. Walsh,et al.  Harnessing the biosynthetic code: combinations, permutations, and mutations. , 1998, Science.

[13]  N. Guex,et al.  SWISS‐MODEL and the Swiss‐Pdb Viewer: An environment for comparative protein modeling , 1997, Electrophoresis.

[14]  C. Khosla,et al.  Malonyl-CoA:ACP transacylase from Streptomyces coelicolor has two alternative catalytically active nucleophiles. , 2001, Biochemistry.

[15]  M. Marahiel,et al.  Crystal structure of the surfactin synthetase‐activating enzyme Sfp: a prototype of the 4′‐phosphopantetheinyl transferase superfamily , 1999, The EMBO journal.

[16]  David O'Hagan,et al.  The polyketide metabolites , 1991 .

[17]  C. Hutchinson,et al.  Characterization of the enzymatic domains in the modular polyketide synthase involved in rifamycin B biosynthesis by Amycolatopsis mediterranei. , 1998, Gene.

[18]  R J Heath,et al.  Identification and Analysis of the Acyl Carrier Protein (ACP) Docking Site on β-Ketoacyl-ACP Synthase III* , 2001, The Journal of Biological Chemistry.

[19]  Z Dauter,et al.  The Escherichia coli Malonyl-CoA:Acyl Carrier Protein Transacylase at 1.5-Å Resolution. , 1995, The Journal of Biological Chemistry.

[20]  K. Parris,et al.  Crystal structures of substrate binding to Bacillus subtilis holo-(acyl carrier protein) synthase reveal a novel trimeric arrangement of molecules resulting in three active sites. , 2000, Structure.

[21]  Tim J. P. Hubbard,et al.  SCOP database in 2002: refinements accommodate structural genomics , 2002, Nucleic Acids Res..

[22]  J. Szulmajster.  Protein folding , 1988, Bioscience reports.

[23]  P. Berna,et al.  Crystal Structure of the Open Form of Dog Gastric Lipase in Complex with a Phosphonate Inhibitor* , 2002, The Journal of Biological Chemistry.

[24]  E A Merritt,et al.  Raster3D: photorealistic molecular graphics. , 1997, Methods in enzymology.

[25]  C R Hutchinson,et al.  Malonyl-coenzyme A:acyl carrier protein acyltransferase of Streptomyces glaucescens: a possible link between fatty acid and polyketide biosynthesis. , 1995, Biochemistry.

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

[27]  D. Cane,et al.  Dissecting the role of acyltransferase domains of modular polyketide synthases in the choice and stereochemical fate of extender units. , 1999, Biochemistry.

[28]  J. Ganguly.  Studies on the mechanism of fatty acid synthesis. VII. Biosynthesis of fatty acids from malonyl CoA. , 1960, Biochimica et biophysica acta.

[29]  John Crosby,et al.  Kinetic and mechanistic analysis of the malonyl CoA:ACP transacylase from Streptomyces coelicolor indicates a single catalytically competent serine nucleophile at the active site. , 2002, Biochemistry.

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

[31]  L. Katz,et al.  Novel macrolides through genetic engineering , 1999, Medicinal research reviews.

[32]  M. Bibb,et al.  Purification of a malonyltransferase from Streptomyces coelicolor A3(2) and analysis of its genetic determinant , 1995, Journal of bacteriology.