title: A novel induced-fit reaction mechanism of asymmetric hot dog thioesterase PAAI.
abstract: Hot dog fold proteins sharing the characteristic "hot dog" fold are known to involve certain coenzyme A binding enzymes with various oligomeric states. In order to elucidate the oligomerization-function relationship of the hot dog fold proteins, crystal structures of the phenylacetate degradation protein PaaI from Thermus thermophilus HB8 (TtPaaI), a tetrameric acyl-CoA thioesterase with the hot dog fold, have been determined and compared with those of other family members. In the liganded crystal forms with coenzyme A derivatives, only two of four intersubunit catalytic pockets of the TtPaaI tetramer are occupied by the ligands. A detailed structural comparison between several liganded and unliganded forms reveals that a subtle rigid-body rearrangement of subunits within 2 degrees upon binding of the first two ligand molecules can induce a strict negative cooperativity to prevent further binding at the remaining two pockets, indicating that the so-called "half-of-the-sites reactivity" of oligomeric enzymes is visualized for the first time. Considering kinetic and mutational analyses together, a possible reaction mechanism of TtPaaI is proposed; one tetramer binds only two acyl-CoA molecules with a novel asymmetric induced-fit mechanism and carries out the hydrolysis according to a base-catalyzed reaction through activation of a water molecule by Asp48. From a structural comparison with other family members, it is concluded that a subgroup of the hot dog fold protein family, referred to as "asymmetric hot dog thioesterases" including medium chain acyl-CoA thioesterase II from Escherichia coli and human thioesterase III, might share the same oligomerization mode and the asymmetric induced-fit mechanism as observed in TtPaaI.
authors.affiliation: Highthroughput Factory, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Sayo-gun, Hyogo 679-5148, Japan. kunisima@spring8.or.jp
authors.forename: Naoki
authors.initials: N
authors.lastname: Kunishima
authors.forename: Yukuhiko
authors.initials: Y
authors.lastname: Asada
authors.forename: Mayumi
authors.initials: M
authors.lastname: Sugahara
authors.forename: Jun
authors.initials: J
authors.lastname: Ishijima
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Nodake
authors.forename: Mitsuaki
authors.initials: M
authors.lastname: Sugahara
authors.forename: Masashi
authors.initials: M
authors.lastname: Miyano
authors.forename: Seiki
authors.initials: S
authors.lastname: Kuramitsu
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.forename: Michihiro
authors.initials: M
authors.lastname: Sugahara
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010170:Palmitoyl-CoA Hydrolase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D010170:Palmitoyl-CoA Hydrolase