High-resolution studies of hydride transfer in the ferredoxin:NADP reductase superfamily.

Unlabelled: Ferredoxin: NADP reductase (FNR) is an FAD-containing enzyme best known for catalysing the transfer of electrons from ferredoxin (Fd) to NADP to make NADPH during photosynthesis. It is also the prototype for a broad enzyme superfamily, including the NADPH oxidases (NOXs) that all catalyse similar FAD-enabled electron transfers between NAD(P)H and one-electron carriers. Here, we define further mechanistic details of the NAD(P)H ⇌ FAD hydride-transfer step of the reaction based on spectroscopic studies and high-resolution (~ 1.

Functional role of a conserved arginine residue located on a mobile loop of alkanesulfonate monooxygenase.

The structure of the flavin-dependent alkanesulfonate monooxygenase (SsuD) exists as a TIM-barrel structure with an insertion region located over the active site that contains a conserved arginine (Arg297) residue present in all SsuD homologues. Substitution of Arg297 with alanine (R297A SsuD) or lysine (R297K SsuD) was performed to determine the functional role of this conserved residue in SsuD catalysis. While the more conservative R297K SsuD possessed a lower k(cat)/K(m) value (0.

Catalytic role of a conserved cysteine residue in the desulfonation reaction by the alkanesulfonate monooxygenase enzyme.

Detailed kinetic studies were performed in order to determine the role of the single cysteine residue in the desulfonation reaction catalyzed by SsuD. Mutation of the conserved cysteine at position 54 in SsuD to either serine or alanine had little effect on FMNH(2) binding. The k(cat)/K(m) value for the C54S SsuD variant increased 3-fold, whereas the k(cat)/K(m) value for C54A SsuD decreased 6-fold relative to wild-type SsuD.

Catalytic importance of the substrate binding order for the FMNH2-dependent alkanesulfonate monooxygenase enzyme.

The two-component alkanesulfonate monooxygenase system from Escherichia coli includes an FMN reductase (SsuE) and an FMNH2-dependent alkanesulfonate monooxygenase (SsuD) involved in the acquisition of sulfur from alkanesulfonates during sulfur starvation. The SsuD enzyme directly catalyzes the oxidation of alkanesulfonate to aldehyde and sulfite in the presence of O2 and FMNH2. The goal of these studies was to investigate the kinetic mechanism of SsuD through rapid reaction kinetics and substrate binding studies.

A general synthesis of tris-indole derivatives as potential iron chelators.

The development of a novel route for the synthesis of a new class of compounds is described. The first tripodal, tris-indole amines are prepared by straightforward routes.