title: A new type of dihydroorotate dehydrogenase, type 1S, from the thermoacidophilic archaeon Sulfolobus solfataricus.
abstract: Dihydroorotate dehydrogenase (DHOD) (EC 1.3.3.1) from the thermoacidophilic archaeon Sulfolobus solfataricus P2 (DSM 1617) was partially purified 3,158-fold, characterized, and the encoding genes identified. Based on enzymological as well as phylogenetic methods, dihydroorotate dehydrogenase from S. solfataricus (DHODS) represents a new type of DHOD, type 1S. Furthermore, it is unable to use any of the (type-specific) natural electron acceptors employed by all other presently known DHODs. DHODS shows optimal activity at 70 degrees C in the pH range 7-8.5. It is capable of using ferricyanide, 2,6-dichlorophenolindophenol (DCIP), Q(0), and molecular oxygen as electron acceptor. Kinetic studies employing ferricyanide indicate a two-site ping-pong mechanism with K(M) values of 44.2+/-1.9 microM for the substrate dihydroorotate and 344+/-21 microM for the electron acceptor ferricyanide, as well as competitive product inhibition with a K(i) of 23.7+/-3.4 microM for the product orotate (OA). The specific activity, as determined from a partially purified sample, is approximately 20 micromol mg(-1) min(-1). DHODS is a heteromeric enzyme comprising a catalytic subunit encoded by pyrD (291 aa; MW=31.1 kDa) and an electron acceptor subunit (208 aa; MW=23.6 kDa), encoded by orf1. DHODS employs a serine as catalytic base, which is unique for a cytosolic DHOD. To our knowledge, this work represents not only the first study on an archaeal DHOD but the first on a nonmesophilic DHOD as well.
authors.affiliation: Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, Sølvgade 83 H, 1307 Copenhagen K, Denmark.
authors.forename: Gravgaard
authors.initials: G
authors.lastname: Sørensen
authors.forename: Gert
authors.initials: G
authors.lastname: Dandanell
mesh_terms: D000090722:Dihydroorotate Dehydrogenase; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology*
chemical_list: D000090722:Dihydroorotate Dehydrogenase; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors