Steady-State Kinetics and Spectroscopic Characterization of Enzyme–tRNA Interactions for the Non-Heme Diiron tRNA-Monooxygenase, MiaE

• Published in: Biochemistry (Easton) 2015-01, Vol.54 (2), p.363-376
• Main Authors: Subedi, Bishnu P, Corder, Andra L, Zhang, Siai, Foss, Frank W, Pierce, Brad S
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Circular Dichroism; Electron Spin Resonance Spectroscopy; Kinetics; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - metabolism; Protein Binding; Protein Conformation; RNA, Transfer - metabolism; Salmonella Infections - microbiology; Salmonella typhimurium; Salmonella typhimurium - chemistry; Salmonella typhimurium - enzymology; Salmonella typhimurium - metabolism; Spectroscopy, Mossbauer
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi5012207

MiaE [2-methylthio-N 6-isopentenyl-adenosine(37)-tRNA monooxygenase] isolated from Salmonella typhimurium is a unique non-heme diiron enzyme that catalyzes the O2-dependent post-transcriptional allylic hydroxylation of a hypermodified nucleotide (ms2i6A37) at position 37 of selected tRNA molecules to produce 2-methylthio-N 6-(4-hydroxyisopentenyl)-adenosine(37). In this work, isopentenylated tRNA substrates for MiaE were produced from small RNA oligomers corresponding to the anticodon stem loop (ACSL) region of tRNATrp using recombinant MiaA and dimethylallyl pyrophosphate. Steady-state rates for MiaE-catalyzed substrate hydroxylation were determined using recombinant ferredoxin (Fd) and ferredoxin reductase (FdR) to provide a catalytic electron transport chain (ETC) using NADPH as the sole electron source. As with previously reported peroxide-shunt assays, steady-state product formation retains nearly stoichiometric (>98%) E stereoselectivity. MiaE-catalyzed i6A-ACSLTrp hydroxylation follows Michaelis–Menten saturation kinetics with k cat, K M, and V/K determined to be 0.10 ± 0.01 s–1, 9.1 ± 1.5 μM, and ∼11000 M–1 s–1, respectively. While vastly slower, MiaE-catalyzed hydroxylation of free i6A nucleoside could also be observed using the (Fd/FdR)–ETC assay. By comparison to the V/K determined for i6A-ACSL substrates, an ∼6000-fold increase in enzymatic efficiency is imparted by ACSLTrp–MiaE interactions. The impact of substrate tRNA–MiaE interactions on protein secondary structure and active site electronic configuration was investigated using circular dichroism, dual-mode X-band electron paramagnetic resonance, and Mössbauer spectroscopies. These studies demonstrate that binding of tRNA to MiaE induces a protein conformational change that influences the electronic structure of the diiron site analogous to what has been observed for various bacterial multicomponent diiron monooxygenases upon titration with their corresponding effector proteins. These observations suggest that substrate–enzyme interactions may play a pivotal role in modulating the reactivity of the MiaE diiron active site. Moreover, the simplified monomeric (α) protein configuration exhibited by MiaE provide an unparalleled opportunity to study the impact of protein–effector interactions on non-heme diiron site geometry and reactivity.