Biochemical and Structural Studies of TGT and MiaA: Key Enzymes Involved in Two Types of Hypermodifications
Part II. Hypermodification of base adenosine at position 37. Hypermodification of A37 (position 3' adjacent to the anticodon region) starts with the formation of (isopentenyl)-adenosine (i6A) and in E. coli, the first step is catalyzed by the enzyme MiaA. Through BLAST search, a few protein sequences highly homologous to MiaA were identified. These protein-encoding genes were cloned, over-expressed and purified. Enzymatic assays were carried out to test the putative enzymes but no activities were detected. The failure to detect activities might result from incorrect assay conditions or substrates and more data is yet to be acquired on this aspect. Crystals of the P. aeruginosa protein were obtained and its structure was determined at 2.2 A. Although the electron density of a segment of the protein was not observed, the partial structure nevertheless shows a central channel composed of positive charged residues. A hypothesis of the MiaA catalytic mechanism is proposed.
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign