Mechanism for multiple‐substrates recognition of α‐aminoadipate aminotransferase from Thermus thermophilus

α‐Aminoadipate aminotransferase (AAA‐AT), a homolog of mammalian kynurenine aminotransferase II (Kat II), transfers an amino group to 2‐oxoadipate to yield α‐aminoadipate in lysine biosynthesis through the α‐aminoadipate pathway in Thermus thermophilus. AAA‐AT catalyzes transamination against various substrates, including AAA, glutamate, leucine, and aromatic amino acids. To elucidate the structural change for recognition of various substrates, we determined crystal structures of AAA‐AT in four forms: with pyridoxal 5′‐phosphate (PLP) (PLP complex), with PLP and leucine (PLP/Leu complex), with N‐phosphopyridoxyl‐leucine (PPL) (PPL complex), and with N‐phosphopyridoxyl‐α‐aminoadipate (PPA) at 2.67, 2.26, 1.75, and 1.67 Å resolution, respectively. The PLP complex is in an open state, whereas PLP/Leu, PPL, and PPA complexes are in closed states with maximal displacement (over 7 Å) of the α2 helix and the β1 strand in the small domain to cover the active site, indicating that conformational change is induced by substrate binding. In PPL and PLP/Leu complexes, several hydrophobic residues on the α2 helix recognize the hydrophobic side chain of the bound leucine moiety whereas, in the PPA complex, the α2 helix rotates to place the guanidium moiety of Arg23 on the helix at the appropriate position to interact with the carboxyl side chain of the AAA moiety. These results indicate that AAA‐AT can recognize various kinds of substrates using the mobile α2 helix. The crystal structures and site‐directed mutagenesis revealed that intersubunit‐electrostatic interactions contribute to the elevated thermostability of this enzyme. Proteins 2009. © 2008 Wiley‐Liss, Inc.

[1]  M. Nishiyama,et al.  alpha-Aminoadipate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus. , 2004, Microbiology.

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

[3]  H. Ikushiro,et al.  Binding of C5-dicarboxylic substrate to aspartate aminotransferase: implications for the conformational change at the transaldimination step. , 2005, Biochemistry.

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

[5]  H. Hayashi,et al.  Structures of Escherichia coli branched-chain amino acid aminotransferase and its complexes with 4-methylvalerate and 2-methylleucine: induced fit and substrate recognition of the enzyme. , 2001, Biochemistry.

[6]  J. K. Bhattacharjee,et al.  A unique fungal lysine biosynthesis enzyme shares a common ancestor with tricarboxylic acid cycle and leucine biosynthetic enzymes found in diverse organisms , 1998, Journal of Molecular Evolution.

[7]  Adam Godzik,et al.  Contribution of electrostatic interactions, compactness and quaternary structure to protein thermostability: lessons from structural genomics of Thermotoga maritima. , 2006, Journal of molecular biology.

[8]  M. Nishiyama,et al.  Aspartate Kinase-Independent Lysine Synthesis in an Extremely Thermophilic Bacterium, Thermus thermophilus: Lysine Is Synthesized via α-Aminoadipic Acid Not via Diaminopimelic Acid , 1999, Journal of bacteriology.

[9]  S. Vissers,et al.  Characterisation of Saccharomyces cerevisiae ARO8 and ARO9 genes encoding aromatic aminotransferases I and II reveals a new aminotransferase subfamily , 1998, Molecular and General Genetics MGG.

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

[11]  J. Jansonius,et al.  Crystal structures of Escherichia coli aspartate aminotransferase in two conformations. Comparison of an unliganded open and two liganded closed forms. , 1994, Journal of molecular biology.

[12]  S L Mowbray,et al.  Planar stacking interactions of arginine and aromatic side-chains in proteins. , 1994, Journal of molecular biology.

[13]  H Hayashi,et al.  Crystal structures of Paracoccus denitrificans aromatic amino acid aminotransferase: a substrate recognition site constructed by rearrangement of hydrogen bond network. , 1998, Journal of molecular biology.

[14]  I. Miyahara,et al.  Conformational Change in Aspartate Aminotransferase on Substrate Binding Induces Strain in the Catalytic Group and Enhances Catalysis* , 2003, The Journal of Biological Chemistry.

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

[16]  C. Parsons,et al.  Novel systemically active antagonists of the glycine site of the N-methyl-D-aspartate receptor: electrophysiological, biochemical and behavioral characterization. , 1997, The Journal of pharmacology and experimental therapeutics.

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

[18]  M. Grenson,et al.  Phenylalanine- and tyrosine-auxotrophic mutants of Saccharomyces cerevisiae impaired in transamination , 1998, Molecular and General Genetics MGG.

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

[20]  Harry P. Broquist,et al.  [157] Lysine biosynthesis (yeast) , 1971 .

[21]  S. Kuramitsu,et al.  The Novel Substrate Recognition Mechanism Utilized by Aspartate Aminotransferase of the Extreme Thermophile Thermus thermophilus HB8* , 1998, The Journal of Biological Chemistry.

[22]  W. Gu,et al.  Evolutionary recruitment of biochemically specialized subdivisions of Family I within the protein superfamily of aminotransferases , 1996, Journal of bacteriology.

[23]  F. Rossi,et al.  Crystal Structure of Human Kynurenine Aminotransferase II, a Drug Target for the Treatment of Schizophrenia* , 2008, Journal of Biological Chemistry.

[24]  Jianyong Li,et al.  Crystal Structure of Human Kynurenine Aminotransferase II* , 2008, Journal of Biological Chemistry.

[25]  H. Hayashi,et al.  The imine-pyridine torsion of the pyridoxal 5'-phosphate Schiff base of aspartate aminotransferase lowers its pKa in the unliganded enzyme and is crucial for the successive increase in the pKa during catalysis. , 1998, Biochemistry.

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

[27]  S. Kanaya,et al.  Crystal structure of a human kynurenine aminotransferase II homologue from Pyrococcus horikoshii OT3 at 2.20 Å resolution , 2005, Proteins.

[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]  I. Miyahara,et al.  Crystal structures of branched-chain amino acid aminotransferase complexed with glutamate and glutarate: true reaction intermediate and double substrate recognition of the enzyme. , 2003, Biochemistry.

[31]  A M Lesk,et al.  Domain closure in mitochondrial aspartate aminotransferase. , 1992, Journal of molecular biology.

[32]  A. Ikai,et al.  Branched-chain amino acid aminotransferase of Escherichia coli: overproduction and properties. , 1988, Journal of biochemistry.

[33]  M. Nishiyama,et al.  Characterization of a lysK gene as an argE homolog in Thermus thermophilus HB27 , 2002, FEBS letters.

[34]  M. Nishiyama,et al.  Functional and Evolutionary Relationship between Arginine Biosynthesis and Prokaryotic Lysine Biosynthesis through α-Aminoadipate , 2001 .

[35]  M. Nishiyama,et al.  Characterization of bacterial homocitrate synthase involved in lysine biosynthesis , 2002, FEBS letters.

[36]  S. Erhardt,et al.  Kynurenic acid and schizophrenia. , 2003, Advances in experimental medicine and biology.

[37]  R. Schwarcz,et al.  Endogenous kynurenate controls the vulnerability of striatal neurons to quinolinate: Implications for Huntington's disease , 2006, Experimental Neurology.

[38]  P. Malherbe,et al.  Cloning and Functional Expression of a Soluble Form of Kynurenine/α -Aminoadipate Aminotransferase from Rat Kidney * , 1995, The Journal of Biological Chemistry.

[39]  M. Nishiyama,et al.  Characterization of Homoisocitrate Dehydrogenase Involved in Lysine Biosynthesis of an Extremely Thermophilic Bacterium,Thermus thermophilus HB27, and Evolutionary Implication of β-Decarboxylating Dehydrogenase* , 2003, The Journal of Biological Chemistry.

[40]  M. Nishiyama,et al.  A prokaryotic gene cluster involved in synthesis of lysine through the amino adipate pathway: a key to the evolution of amino acid biosynthesis. , 1999, Genome research.

[41]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[42]  H. Vogel.  Distribution of Lysine Pathways Among Fungi: Evolutionary Implications , 1964, The American Naturalist.

[43]  R. Schwarcz,et al.  The Brain Metabolite Kynurenic Acid Inhibits α7 Nicotinic Receptor Activity and Increases Non-α7 Nicotinic Receptor Expression: Physiopathological Implications , 2001, The Journal of Neuroscience.

[44]  I. Miyahara,et al.  Structure of Thermus thermophilus HB8 aspartate aminotransferase and its complex with maleate. , 1999, Biochemistry.

[45]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[46]  Sudhir Kumar,et al.  MEGA2: molecular evolutionary genetics analysis software , 2001, Bioinform..