title: Mechanism of substrate recognition and insight into feedback inhibition of homocitrate synthase from Thermus thermophilus.
abstract: Homocitrate synthase (HCS) catalyzes aldol-type condensation of acetyl coenzyme A (acetyl-CoA) and alpha-ketoglutarate (alpha-KG) to synthesize homocitrate (HC), which is the first and committed step in the lysine biosynthetic pathway through alpha-aminoadipate. As known in most enzymes catalyzing the first reactions in amino acid biosynthetic pathways, HCS is regulated via feedback inhibition by the end product, lysine. Here, we determined the crystal structures of HCS from Thermus thermophilus complexed with alpha-KG, HC, or lysine. In the HC complex, the C1-carboxyl group of HC, which is derived from acetyl-CoA, is hydrogen-bonded with His-292* from another subunit (indicated by the asterisk), indicating direct involvement of this residue in the catalytic mechanism of HCS. The crystal structure of HCS complexed with lysine showed that lysine is bound to the active site with rearrangement of amino acid residues in the substrate-binding site, which accounts for the competitive inhibition by lysine with alpha-KG. Comparison between the structures suggests that His-72, which is conserved in lysine-sensitive HCSs and binds the C5-carboxyl group of alpha-KG, serves as a switch for the conformational change. Replacement of His-72 by leucine made HCS resistant to lysine inhibition, demonstrating the regulatory role of this conserved residue.
authors.affiliation: From the Biotechnology Research Center, University of Tokyo, Tokyo 113-8657 and.
authors.forename: Takuya
authors.initials: T
authors.lastname: Okada
authors.affiliation: From the Biotechnology Research Center, University of Tokyo, Tokyo 113-8657 and.
authors.forename: Takeo
authors.initials: T
authors.lastname: Tomita
authors.affiliation: From the Biotechnology Research Center, University of Tokyo, Tokyo 113-8657 and.
authors.forename: Asri P
authors.initials: AP
authors.lastname: Wulandari
authors.affiliation: From the Biotechnology Research Center, University of Tokyo, Tokyo 113-8657 and.
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Kuzuyama
authors.affiliation: From the Biotechnology Research Center, University of Tokyo, Tokyo 113-8657 and; the RIKEN SPring-8 Center, Hyogo 679-5148, Japan. Electronic address: umanis@mail.ecc.u-tokyo.ac.jp.
authors.forename: Makoto
authors.initials: M
authors.lastname: Nishiyama
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D025461:Feedback, Physiological; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D007656:Ketoglutaric Acids / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D007652:Oxo-Acid-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics; D014233:Tricarboxylic Acids / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007656:Ketoglutaric Acids; D014233:Tricarboxylic Acids; C028143:homocitric acid; D006639:Histidine; C021365:homocitrate synthase; D007652:Oxo-Acid-Lyases; D007930:Leucine; D008239:Lysine