title: A detailed biochemical characterization of phosphopantothenate synthetase, a novel enzyme involved in coenzyme A biosynthesis in the Archaea.
abstract: We have previously reported that the majority of the archaea utilize a novel pathway for coenzyme A biosynthesis (CoA). Bacteria/eukaryotes commonly use pantothenate synthetase and pantothenate kinase to convert pantoate to 4'-phosphopantothenate. However, in the hyperthermophilic archaeon Thermococcus kodakarensis, two novel enzymes specific to the archaea, pantoate kinase and phosphopantothenate synthetase, are responsible for this conversion. Here, we examined the enzymatic properties of the archaeal phosphopantothenate synthetase, which catalyzes the ATP-dependent condensation of 4-phosphopantoate and β-alanine. The activation energy of the phosphopantothenate synthetase reaction was 82.3 kJ mol(-1). In terms of substrate specificity toward nucleoside triphosphates, the enzyme displayed a strict preference for ATP. Among several amine substrates, activity was detected with β-alanine, but not with γ-aminobutyrate, glycine nor aspartate. The phosphopantothenate synthetase reaction followed Michaelis-Menten kinetics toward β-alanine, whereas substrate inhibition was observed with 4-phosphopantoate and ATP. Feedback inhibition by CoA/acetyl-CoA and product inhibition by 4'-phosphopantothenate were not observed. By contrast, the other archaeal enzyme pantoate kinase displayed product inhibition by 4-phosphopantoate in a non-competitive manner. Based on our results, we discuss the regulation of CoA biosynthesis in the archaea.
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, 615-8510, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Ishibashi
authors.forename: Hiroya
authors.initials: H
authors.lastname: Tomita
authors.forename: Yuusuke
authors.initials: Y
authors.lastname: Yokooji
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Morikita
authors.forename: Bunta
authors.initials: B
authors.lastname: Watanabe
authors.forename: Jun
authors.initials: J
authors.lastname: Hiratake
authors.forename: Asako
authors.initials: A
authors.lastname: Kishimoto
authors.forename: Akiko
authors.initials: A
authors.lastname: Kita
authors.forename: Kunio
authors.initials: K
authors.lastname: Miki
authors.forename: Tadayuki
authors.initials: T
authors.lastname: Imanaka
authors.forename: Haruyuki
authors.initials: H
authors.lastname: Atomi
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003065:Coenzyme A / Q000096:biosynthesis*; D007700:Kinetics; D010205:Pantothenic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000138:chemical synthesis; D010453:Peptide Synthases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D019712:Thermococcus / Q000201:enzymology*
chemical_list: D019843:Archaeal Proteins; D010710:Phosphates; D010205:Pantothenic Acid; C011988:phosphopantothenic acid; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C023088:pantothenate kinase; D010453:Peptide Synthases; D000409:Alanine; D003065:Coenzyme A