Structural bioinformatics analysis of enzymes involved in the biosynthesis pathway of the hypermodified nucleoside ms2io6A37 in tRNA

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Abstract
TRNAs from all organisms contain posttranscriptionally modified nucleosides, which are derived from the four canonical nucleosides. In most tRNAs that read codons beginning with U, adenosine in the position 37 adjacent to the 3′ position of the anticodon is modified to N6‐(Δ2‐isopentenyl) adenosine (i6A). In many bacteria, such as Escherichia coli, this residue is typically hypermodified to N6‐isopentenyl‐2‐thiomethyladenosine (ms2i6A). In a few bacteria, such as Salmonella typhimurium, ms2i6A can be further hydroxylated to N6‐(cis‐4‐hydroxyisopentenyl)‐2‐thiomethyladenosine (ms2io6A). Although the enzymes that introduce the respective modifications (prenyltransferase MiaA, methylthiotransferase MiaB, and hydroxylase MiaE) have been identified, their structures remain unknown and sequence‐function relationships remain obscure. We carried out sequence analysis and structure prediction of MiaA, MiaB, and MiaE, using the protein fold‐recognition approach. Three‐dimensional models of all three proteins were then built using a new modeling protocol designed to overcome uncertainties in the alignments and divergence between the templates. For MiaA and MiaB, the catalytic core was built based on the templates from the P‐loop NTPase and Radical‐SAM superfamilies, respectively. For MiaB, we have also modeled the C‐terminal TRAM domain and the newly predicted N‐terminal flavodoxin‐fold domain. For MiaE, we confidently predict that it shares the three‐dimensional fold with the ferritin‐like four‐helix bundle proteins and that it has a similar active site and mechanism of action to diiron carboxylate enzymes, in particular, methane monooxygenase (E.C.1.14.13.25) that catalyses the biological hydroxylation of alkanes. Our models provide the first structural platform for enzymes involved in the biosynthesis of i6A, ms2i6A, and ms2io6A, explain the data available from the literature and will help to design further experiments and interpret their results. Proteins 2008.