Structural Insight into concerted inhibition of alpha 2 beta 2-type aspartate kinase from Corynebacterium glutamicum.

Aspartate kinase (AK) catalyzes the first step of the biosynthesis of the aspartic acid family amino acids, and is regulated via feedback inhibition by end-products including Thr and Lys. To elucidate the mechanism of this inhibition, we determined the crystal structure of the regulatory subunit of AK from Corynebacterium glutamicum at 1.58 A resolution in the Thr-binding form, the first crystal structure of the regulatory subunit of alpha(2)beta(2)-type AK. The regulatory subunit contains two ACT domain motifs per monomer and is arranged as a dimer. Two non-equivalent ACT domains from different chains form an effector-binding unit that binds a single Thr molecule, and the resulting two effector-binding units of the dimer associate perpendicularly in a face-to-face manner. The regulatory subunit is a monomer in the absence of Thr but becomes a dimer by adding Thr. The dimerization is eliminated in mutant AKs with changes in the Thr-binding region, suggesting that the dimerization induced by Thr binding is a key step in the inhibitory mechanism of AK from C. glutamicum. A putative Lys-binding site and the inhibitory mechanism of CgAK are discussed.

[1]  D. Stammers,et al.  Structures of R- and T-state Escherichia coli Aspartokinase III , 2006, Journal of Biological Chemistry.

[2]  J. Ferrer,et al.  A Novel Organization of ACT Domains in Allosteric Enzymes Revealed by the Crystal Structure of Arabidopsis Aspartate Kinase[W] , 2006, The Plant Cell Online.

[3]  C. Marco-Marín,et al.  Site-directed mutagenesis of Escherichia coli acetylglutamate kinase and aspartokinase III probes the catalytic and substrate-binding mechanisms of these amino acid kinase family enzymes and allows three-dimensional modelling of aspartokinase. , 2003, Journal of molecular biology.

[4]  D E McRee,et al.  XtalView/Xfit--A versatile program for manipulating atomic coordinates and electron density. , 1999, Journal of structural biology.

[5]  J. Sacchettini,et al.  Crystal Structure of ATP Phosphoribosyltransferase fromMycobacterium tuberculosis * , 2003, The Journal of Biological Chemistry.

[6]  B. Miflin,et al.  S-adenosylmethionine—a novel regulator of aspartate kinase , 1980, Nature.

[7]  G. A. Grant.  The ACT Domain: A Small Molecule Binding Domain and Its Role as a Common Regulatory Element* , 2006, Journal of Biological Chemistry.

[8]  S. Black,et al.  beta-Aspartokinase and beta-aspartyl phosphate. , 1955, The Journal of biological chemistry.

[9]  J. Kalinowski,et al.  Aspartokinase genes lysCα and lysCβ overlap and are adjacent to the aspartate β-semialdehyde dehydrogenase gene asd in Corynebacterium glutamicum , 1990, Molecular and General Genetics MGG.

[10]  Gilles Curien,et al.  Mechanism of Control of Arabidopsis thaliana Aspartate Kinase-Homoserine Dehydrogenase by Threonine* , 2003, The Journal of Biological Chemistry.

[11]  R. Dumas,et al.  Evidence for two distinct effector-binding sites in threonine deaminase by site-directed mutagenesis, kinetic, and binding experiments. , 2000, Biochemistry.

[12]  M. Vyazmensky,et al.  Structure of the regulatory subunit of acetohydroxyacid synthase isozyme III from Escherichia coli. , 2006, Journal of molecular biology.

[13]  I. Shiio,et al.  Concerted inhibition and its reversal by end products of aspartate kinase in Brevibacterium flavum. , 1969, Journal of biochemistry.

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

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

[16]  M. Nishiyama,et al.  An operon encoding aspartokinase and purine phosphoribosyltransferase in Thermus flavus. , 1995, Microbiology.

[17]  Patrice Gouet,et al.  ESPript: analysis of multiple sequence alignments in PostScript , 1999, Bioinform..

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

[19]  R. Viola,et al.  The initial step in the archaeal aspartate biosynthetic pathway catalyzed by a monofunctional aspartokinase. , 2006, Acta crystallographica. Section F, Structural biology and crystallization communications.

[20]  H. Paulus,et al.  Nucleotide sequence of the overlapping genes for the subunits of Bacillus subtilis aspartokinase II and their control regions. , 1987, The Journal of biological chemistry.

[21]  Robert T Sauer,et al.  Crystal structure of the nickel-responsive transcription factor NikR , 2003, Nature Structural Biology.

[22]  H. Sahm,et al.  Genetic and biochemical analysis of the aspartokinase from Corynebacterium glutamicum , 1991, Molecular microbiology.

[23]  F. Gil-Ortiz,et al.  Structure of acetylglutamate kinase, a key enzyme for arginine biosynthesis and a prototype for the amino acid kinase enzyme family, during catalysis. , 2002, Structure.

[24]  Thomas C Terwilliger,et al.  Automated structure solution, density modification and model building. , 2002, Acta crystallographica. Section D, Biological crystallography.

[25]  Takeo Tomita,et al.  Purification, crystallization and preliminary X-ray analysis of the regulatory subunit of aspartate kinase from Thermus thermophilus. , 2007, Acta crystallographica. Section F, Structural biology and crystallization communications.

[26]  E. Koonin,et al.  Gleaning non-trivial structural, functional and evolutionary information about proteins by iterative database searches. , 1999, Journal of molecular biology.

[27]  O. Martínez-Costa,et al.  Characterization of the aspartate kinase from Saccharomyces cerevisiae and of its interaction with threonine. , 2004, Biochemical and biophysical research communications.

[28]  M. Nishiyama,et al.  Conversion of feedback regulation in aspartate kinase by domain exchange. , 2004, Biochemical and biophysical research communications.

[29]  Alexei Vagin,et al.  Structural bases of feed-back control of arginine biosynthesis, revealed by the structures of two hexameric N-acetylglutamate kinases, from Thermotoga maritima and Pseudomonas aeruginosa. , 2006, Journal of molecular biology.

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

[31]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[32]  M. Nishiyama,et al.  Kinetic and mutation analyses of aspartate kinase from Thermus flavus. , 1999, Journal of bioscience and bioengineering.

[33]  D. J. Schuller,et al.  The allosteric ligand site in the Vmax-type cooperative enzyme phosphoglycerate dehydrogenase , 1995, Nature Structural Biology.

[34]  G. Gilliland,et al.  Structure and control of pyridoxal phosphate dependent allosteric threonine deaminase. , 1998, Structure.

[35]  D. Chipman,et al.  The ACT domain family. , 2001, Current opinion in structural biology.

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

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

[38]  J. Kalinowski,et al.  Cloning of a DNA fragment fromCorynebacterium glutamicum conferring aminoethyl cysteine resistance and feedback resistance to aspartokinase , 2004, Applied Microbiology and Biotechnology.

[39]  H. E. Umbarger,et al.  Amino acid biosynthesis and its regulation. , 1978, Annual review of biochemistry.