A Non-heme Diiron Monooxygenase From Salmonella Typhimurium: Biochemical Characterization Of TRNA-modifying MiaE
Abstract
MiaE is a carboxylate bridged, non-heme diiron monooxygenase which catalyzes the O2-dependent hydroxylation of a hypermodified tRNA nucleoside at position 37 (2-methylthio-N-6-isopentenyl adenosine (37)-tRNA) [designated ms²i⁶A₃₇] to produce ms²i⁶A₃₇. In Salmonella typhimurium, synthesis of ms²i⁶A₃₇ requires a multi-step enzyme-catalyzed process that includes two other enzymes, MiaA and MiaB. Therefore, to study the hydroxylation step in this reaction pathway, all the elements within this reaction pathway (tRNA substrate, MiaA, MiaB, and MiaE) are required. In this work, methods for in vitro tRNA synthesis were developed for the biosynthesis of viable MiaA/MiaB/MiaE substrates. Additionally, the enzymes MiaA and MiaB were recombinantly cloned, sequence verified, and demonstrated to express soluble maltose-binding protein (MBP) fusion proteins. Recombinant MiaA and MiaE (cloned previously) were demonstrated to be catalytically active.