Dihydropyrimidine Dehydrogenase from Escherichia coli: Evidence for the Catalytic Sequence

Dihydropyrimidine dehydrogenase from Escherichia coli (EcDPD) reduces the 5,6-vinylic bond of pyrimidines using electrons from NADH. Here, we expand on our previous results that demonstrated that EcDPD undergoes reductive activation, that turnover is enhanced in the presence of NADH, and that EcDPD will reoxidize completely in the absence of excess NADH (Alt, T.B. et al. Arch. Biochem. Biophys., 2023, 748, 109772). A linear free energy relationship analysis using halogenated uracils revealed that reduction of the pyrimidine is not rate-limiting in turnover. In addition, mutating the catalytic cysteine to serine (C137S) slowed pyrimidine reduction but did not make it rate-limiting. [4S-²H] NADH kinetic isotope effects (KIE) were observed only for reductive activation. Together, these results demonstrate that electron transmission from the FAD to the FMN is rate-limiting in EcDPD turnover. An NAD⁺·FADH₂ charge transfer absorption showed that the reductively activated enzyme is in the FADH₂·4(Fe₄S₄)·FMN state. The electrostatic environment around the FAD of Sus scrofa DPD (SsDPD) was made more positive by point mutations in an attempt to retain electrons on the FAD; however, the reductively activated state remained unchanged, indicating that this process is controlled by something other than the electrostatics near the FAD cofactor. 5-ethynyluracil inactivation was used as a reporter for the position of the mobile loop containing Cys137, establishing that the loop exists in the "out" position in the oxidized enzyme, but is biased inward upon reductive activation.