MiaE; 2-methylthio-N-6-isopentenyl adenosine-37 (ms2i6A37) tRNA monooxygenase is a monomeric non-heme diiron enzyme which catalyzes the post-transcriptional allylic hydroxylation of transfer RNA. In the resting state, MiaE shows a characteristic absorption band (320-370 nm) which is typical of a non-heme diferric cluster containing a bridging �-oxo ligand. In this work, we utilized various spectroscopic and kinetic methods to study enzyme-substrate interaction and its impact on reactivity. MiaE activity was verified utilizing both synthetically produced isopentenylated nucleosides (i6A) and enzymatically generated isopentenylated RNA oligomers (17 nucleotides) corresponding to the antistem codon loop of tRNAtrp (i6A-ASLtrp). Both peroxide shunt and a catalytic electron transport chain (NADPH, ferredoxin reductase, and ferredoxin) were utilized and compared in steady state assay. As expected, the biological reduction pathway is faster than the peroxide shunt; similarly the rate of isopentenylated RNA oligomer hydroxylation is significantly faster than the free nucleoside. The effect of interaction of enzyme with its substrate analog (ASL) was studied in peripheral as well as in relatively small active site level. Both circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy show a tight binding of MiaE with ASLtrp. The EPR result indicates that the binding of ASLtrp significantly alters the electronic structure and electronic relaxation rates of the reduced MiaE active site.

Kinetic and spectroscopic study of enzyme-substrate interactions in a tRNA monoxoygenase MiaE ( a non-heme diiron enzyme)

247th National Meeting (2014)

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