Dihydropyrmidine dehydrogenase from Escherichia coli: Transient state analysis reveals both reductive activation prior to turnover and diminished substrate effector roles relative to the mammalian form.

Dihydropyrimidine dehydrogenase (DPD) is an enzyme that uses an elaborate architecture to catalyze a simple net reaction: the reduction of the vinylic bond of uracil and thymine. Known DPDs have two active sites separated by approximately 60 Å. One active site has an FAD cofactor and binds NAD(P) and the other has an FMN cofactor and binds pyrimidines. The intervening distance is spanned by four Fe4S4 centers that act as an electron conduit. Recent advancements with porcine DPD have revealed unexpected chemical sequences where the enzyme undergoes reductive activation by transferring two electrons from NADPH to the FMN via the FAD such that the active form has the cofactor set FAD•4(Fe4S4)•FMNH2. Here we describe the first comprehensive kinetic investigation of a bacterial form of DPD. Using primarily transient state methods, DPD from E. coli (EcDPD) was shown to have a similar mechanism to that observed with the mammalian form in that EcDPD is observed to undergo reductive activation before pyrimidine reduction and displays half-of-sites activity. However, two distinct aspects of the EcDPD reaction relative to the mammalian enzyme were observed that relate to the effector roles for substrates: (i) the enzyme will rapidly take up electrons from NADH, reducing a flavin in the absence of pyrimidine substrate, and (ii) the activated form of the enzyme can become fully oxidized by transferring electrons to pyrimidine substrates in the absence of NADH.