title: Structural and functional characterization of 4-hydroxyphenylpyruvate dioxygenase from the thermoacidophilic archaeon Picrophilus torridus.
abstract: 4-Hydroxyphenylpyruvate dioxygenase (Hpd, EC 1.13.11.27) catalyzes the conversion of 4-hydroxyphenylpyruvate into homogentisate in the second step of oxidative tyrosine catabolism. This pathway is known from bacteria and eukaryotes, but so far no archaeal Hpd has been described. Here, we report the biochemical characterization of an Hpd from the extremophilic archaeon Picrophilus torridus (Pt_Hpd), together with its three-dimensional structure at a resolution of 2.6 Å. Two pH optima were observed at 50 °C: pH 4.0 (close to native conditions) and pH 7.0. The enzyme showed only moderate thermostability and was inactivated with a half-life of ~1.5 h even under optimal reaction conditions. At the ideal physiological growth conditions of P. torridus, Pt_Hpd was inactive after 1 h, showing that the enzyme is protected in vivo from denaturation and/or is only partially adapted to the harsh environmental conditions in the cytosol of P. torridus. The influence of different additives on the activity was investigated. Pt_Hpd exhibited a turnover number k(cat) of 9.9 ± 0.6 s(-1) and a substrate binding affinity K(m) of 142 ± 23 µM. In addition, substrate inhibition with a binding affinity K(i) of 1.9 ± 0.3 mM was observed. Pt_Hpd is compared with isoenzymes from other species and the putative bacterial origin of the gene is discussed.
authors.affiliation: Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, 79104, Freiburg, Germany, eduard.frick@pharmazie.uni-freiburg.de.
authors.forename: Eduard
authors.initials: E
authors.lastname: Frick
authors.forename: Thomas
authors.initials: T
authors.lastname: Spatzal
authors.forename: Stefan
authors.initials: S
authors.lastname: Gerhardt
authors.forename: Andreas
authors.initials: A
authors.lastname: Krämer
authors.forename: Oliver
authors.initials: O
authors.lastname: Einsle
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hüttel
mesh_terms: D015111:4-Hydroxyphenylpyruvate Dioxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D011487:Protein Conformation; D019603:Thermoplasmales / Q000201:enzymology*
chemical_list: D019843:Archaeal Proteins; D015111:4-Hydroxyphenylpyruvate Dioxygenase