Crystallographic analysis of the reaction pathway of Zoogloea ramigera biosynthetic thiolase.

Biosynthetic thiolases catalyze the biological Claisen condensation of two acetyl-CoA molecules to form acetoacetyl-CoA. This is one of the fundamental categories of carbon skeletal assembly patterns in biological systems and is the first step in many biosynthetic pathways including those which generate cholesterol, steroid hormones and ketone body energy storage molecules. High resolution crystal structures of the tetrameric biosynthetic thiolase from Zoogloea ramigera were determined (i) in the absence of active site ligands, (ii) in the presence of CoA, and (iii) from protein crystals which were flash frozen after a short soak with acetyl-CoA, the enzyme's substrate in the biosynthetic reaction. In the latter structure, a reaction intermediate was trapped: the enzyme was found to be acetylated at Cys89 and a molecule of acetyl-CoA was bound in the active site pocket. A comparison of the three new structures and the two previously published thiolase structures reveals that small adjustments in the conformation of the acetylated Cys89 side-chain allow CoA and acetyl-CoA to adopt identical modes of binding. The proximity of the acetyl moiety of acetyl-CoA to the sulfur atom of Cys378 supports the hypothesis that Cys378 is important for proton exchange in both steps of the reaction. The thioester oxygen atom of the acetylated enzyme points into an oxyanion hole formed by the nitrogen atoms of Cys89 and Gly380, thus facilitating the condensation reaction. The interaction between the thioester oxygen atom of acetyl-CoA and His348 assists the condensation step of catalysis by stabilizing a negative charge on the thioester oxygen atom. Our structure of acetyl-CoA bound to thiolase also highlights the importance in catalysis of a hydrogen bonding network between Cys89 and Cys378, which includes the thioester oxygen atom of acetyl-CoA, and extends from the catalytic site through the enzyme to the opposite molecular surface. This hydrogen bonding network is different in yeast degradative thiolase, indicating that the catalytic properties of each enzyme may be modulated by differences in their hydrogen bonding networks.

[1]  C. Walsh,et al.  Poly-(R)-3-hydroxybutyrate (PHB) biosynthesis: mechanistic studies on the biological Claisen condensation catalyzed by β-ketoacyl thiolase , 1989 .

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

[3]  V S Lamzin,et al.  The 1.8 A crystal structure of the dimeric peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae: implications for substrate binding and reaction mechanism. , 1997, Journal of molecular biology.

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

[5]  T. Hashimoto,et al.  Medium Chain 3-Ketoacyl-Coenzyme A Thiolase Deficiency: A New Disorder of Mitochondrial Fatty Acid β-Oxidation , 1997, Pediatric Research.

[6]  A. Palotie,et al.  Long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency with the G1528C mutation: clinical presentation of thirteen patients. , 1997, The Journal of pediatrics.

[7]  Y. Modis,et al.  A biosynthetic thiolase in complex with a reaction intermediate: the crystal structure provides new insights into the catalytic mechanism. , 1999, Structure.

[8]  T. Hashimoto,et al.  Characterization of N93S, I312T, and A333P missense mutations in two Japanese families with mitochondrial acetoacetyl‐CoA thiolase deficiency , 1998, Human mutation.

[9]  C. Walsh,et al.  Biosynthetic thiolase from Zoogloea ramigera. Mutagenesis of the putative active-site base Cys-378 to Ser-378 changes the partitioning of the acetyl S-enzyme intermediate. , 1992, The Journal of biological chemistry.

[10]  V S Lamzin,et al.  Automated refinement of protein models. , 1993, Acta crystallographica. Section D, Biological crystallography.

[11]  C. Walsh,et al.  Mechanistic studies on beta-ketoacyl thiolase from Zoogloea ramigera: identification of the active-site nucleophile as Cys89, its mutation to Ser89, and kinetic and thermodynamic characterization of wild-type and mutant enzymes. , 1989, Biochemistry.

[12]  G Vriend,et al.  WHAT IF: a molecular modeling and drug design program. , 1990, Journal of molecular graphics.

[13]  H. Gilbert,et al.  The relation of acyl transfer to the overall reaction of thiolase I from porcine heart. , 1981, The Journal of biological chemistry.

[14]  F. Lynen,et al.  Reinigung und Kristallisation der Thiolase, Untersuchungen zum Wirkungsmechanismus , 1968 .

[15]  C. Walsh,et al.  Bio-Claisen condensation catalyzed by thiolase from Zoogloea ramigera. Active site cysteine residues , 1989 .

[16]  P. Hartlage,et al.  Mitochondrial acetoacetyl-CoA thiolase deficiency. , 1986, Biochemical medicine and metabolic biology.

[17]  C. Walsh,et al.  Biosynthetic thiolase from Zoogloea ramigera. Evidence for a mechanism involving Cys-378 as the active site base. , 1991, The Journal of biological chemistry.

[18]  H. Schulz,et al.  beta-oxidation of fatty acids in mitochondria, peroxisomes, and bacteria: a century of continued progress. , 1995, Progress in lipid research.

[19]  S. Remington,et al.  A very short hydrogen bond provides only moderate stabilization of an enzyme-inhibitor complex of citrate synthase. , 1994, Biochemistry.

[20]  M. Noble,et al.  Structure of triosephosphate isomerase from Escherichia coli determined at 2.6 A resolution. , 1993, Acta crystallographica. Section D, Biological crystallography.

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

[22]  T Hashimoto,et al.  Properties of peroxisomal 3-ketoacyl-coA thiolase from rat liver. , 1981, Journal of biochemistry.

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

[24]  Wolfgang Kabsch,et al.  Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants , 1993 .

[25]  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.