Structural basis for tRNA methylthiolation by the radical SAM enzyme MiaB

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Post-transcriptional modifications of transfer RNAs (tRNAs) are essential for accurate protein synthesis.
• The 2-methylthio-N6-isopentenyladenosine (ms2i6A) modification at tRNA position 37 (A37) enhances translational fidelity by promoting correct codon-anticodon pairing.
• MiaB, a radical S-adenosylmethionine (SAM) methylthiotransferase, catalyzes the installation of ms2i6A onto isopentenyladenosine (i6A).

Purpose of the Study:

• To elucidate the mechanism by which MiaB installs the ms2i6A modification.
• To understand the role of the auxiliary [Fe4S4]aux cluster in sulfur transfer during the modification process.
• To provide structural insights into MiaB function.

Main Methods:

• X-ray crystallography was used to determine the structures of MiaB from Bacteroides uniformis.
• Biochemical assays were employed to investigate the enzymatic mechanism.

Main Results:

• Structural data revealed a two-step mechanism for ms2i6A installation.
• The first step involves methylation of the auxiliary cluster's bridging µ-sulfido ion by one SAM molecule.
• The second step involves reductive cleavage of a second SAM molecule by the radical SAM cluster, followed by hydrogen abstraction from the i6A37 substrate after C2 rehybridization.

Conclusions:

• The study presents a detailed mechanistic model for MiaB-catalyzed ms2i6A modification.
• The findings clarify the role of the auxiliary cluster in sulfur transfer and the radical SAM cluster in C-H bond functionalization.
• This work advances the understanding of enzymatic sulfur incorporation into C-H bonds.