Decoding Substrate Selectivity of an Archaeal RlmCD-like Methyltransferase Through Its Salient Traits

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• 5-Methyluridine (m5U) modifications are crucial for 23S rRNA function in bacteria.
• Gram-negative bacteria use RlmC and RlmD, while Gram-positive bacteria use RlmCD for m5U modification.
• Archaea, particularly Thermococcales, possess genes for archaeal (tRNA and rRNA) m5U (Arm5U) methyltransferases.

Purpose of the Study:

• To structurally and functionally characterize the rRNA-specific Arm5U methyltransferase (PhRlmCD) from Pyrococcus horikoshii.
• To understand the substrate selectivity and catalytic mechanism of PhRlmCD.
• To investigate the evolutionary origin of Arm5U methyltransferases.

Main Methods:

• X-ray crystallography for structural determination of PhRlmCD.
• Biochemical assays to assess methyltransferase activity.
• Bioinformatic analysis to infer evolutionary relationships.

Main Results:

• PhRlmCD exhibits a dynamic hinge movement and unique structural features, including elongated positively charged loops and rotational variations in the TRAM domain.
• These structural characteristics influence substrate selectivity and enable dual MTase activities, similar to bacterial RlmCD.
• The study suggests that mini-rRNA fragments may interact with PhRlmCD to facilitate its dual functions.

Conclusions:

• Arm5U methyltransferases in archaea likely evolved through horizontal gene transfer, primarily from Gram-positive bacteria.
• The structural and functional insights into PhRlmCD provide a deeper understanding of rRNA modification diversity across domains of life.
• This research highlights the adaptability of methyltransferase enzymes and their evolutionary trajectories.