Substrate-binding loop interactions with pseudouridine trigger conformational changes that promote catalytic...

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Pseudouridine is a major RNA modification crucial for cellular function.
• Pseudouridine kinase (PUKI) catalyzes the initial step in pseudouridine catabolism, phosphorylating it to pseudouridine 5'-monophosphate.
• The molecular mechanism underlying PUKI's high selectivity for pseudouridine over uridine remains unclear.

Purpose of the Study:

• To elucidate the structural and molecular basis for PUKI's substrate selectivity.
• To investigate the role of specific amino acid residues in PUKI's catalytic mechanism.

Main Methods:

• X-ray crystallography to determine EcPUKI structures in various ligation states.
• Kinetic and fluorescence spectroscopy analyses.
• Site-directed mutagenesis (S30A) to probe functional significance.

Main Results:

• EcPUKI shares structural similarity with other PUKI enzymes, featuring an α/β core and a β-stranded small domain.
• Ser30 in the substrate-binding loop interacts with the N1 atom of pseudouridine, inducing conformational changes.
• This Ser30-mediated interaction is critical for conformational changes and subsequent catalysis, as evidenced by S30A mutation and homologous nucleoside binding studies.

Conclusions:

• EcPUKI exhibits pseudouridine-dependent conformational changes.
• The interaction between Ser30 and the pseudouridine N1 atom is essential for PUKI's catalytic activity and selectivity.
• These findings provide structural and functional insights into the high-fidelity catalysis of pseudouridine by PUKI.