Biochemical characterization of rhodoquinone biosynthesis enzyme (RquA)

Abstract

Biological isoprenoid quinones – ubiquinone (UQ), menaquinone (MK), and rhodoquinone (RQ) – play a critical role in cellular bioenergetics by facilitating electron transport in the electron transport chain (ETC). In aerobic respiration, UQ primarily delivers electrons to complex III, with oxygen serving as the final electron acceptor at complex IV. In contrast, under anaerobic conditions, RQ and MK transfer electrons to complex II, where fumarate acts as the terminal electron acceptor, making their biosynthesis essential for organisms that undergo fumarate respiration. RQ is structurally more similar to UQ than MK and is synthesized from UQ in bacteria. The rhodoquinone biosynthesis enzyme (RquA), a member of the S-adenosyl-L-methionine (SAM)-dependent methyltransferase family, was previously identified as being required for RQ biosynthesis in Rhodospirillum rubrum. In this thesis, I employed biophysical, biochemical, and computational approaches to characterize the structural features and functional activity of RquA. Recombinant RquA was purified in the presence of detergents, and an in vitro functional assay was established to assess its enzymatic activity. RquA utilizes SAM as an amino group donor and requires divalent metal cations to convert UQ to RQ. The products formed from the RquA reactions were identified using isotopically labeled substrates and nuclear magnetic resonance (NMR) spectroscopy. Isothermal titration calorimetry (ITC) was used to investigate metal binding, which revealed that Mn²⁺ and Co²⁺ bind RquA with sub-micromolar affinities in a 1:1 stoichiometry. Additionally, starting with an AlphaFold3-derived structural model of RquA, I performed molecular dynamics simulations and mutagenesis studies, revealing that RquA likely functions as a monotopic membrane protein. Membrane association is mediated via amphipathic regions of RquA, where electrostatic interactions involving arginine residues and van der Waals interactions contributed by tryptophan and phenylalanine residues stabilize its binding to the lipid bilayer. This membrane interaction was found to be essential for UQ binding and RquA catalytic activity. Furthermore, I assessed the activity of RquA variants to validate the predicted SAM and Mn²⁺ binding sites within RquA, confirming its role as a novel, non-methylating enzyme related to SAM-dependent methyltransferases. Instead of catalyzing methyl transfer, RquA uniquely transfers the α-amino group from SAM to UQ, defining a previously unrecognized enzymatic mechanism. Together, these findings provide new insights into the molecular basis of RQ biosynthesis and expand our understanding of non-canonical SAM-dependent enzymatic reactions.