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°C in the pH range 7–8.5. It is capable of using ferricyanide, 2,6-dichlorophenolindophenol (DCIP), Q₀, 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 µM for the substrate dihydroorotate and 344±21 µM for the electron acceptor ferricyanide, as well as competitive product inhibition with a K _i of 23.7±3.4 µM for the product orotate (OA). The specific activity, as determined from a partially purified sample, is approximately 20 µmol 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.