Structure and function of the pseudouridine 5’-monophosphate glycosylase PUMY from Arabidopsis thaliana

ABSTRACT

Pseudouridine is a noncanonical C-nucleoside containing a C-C glycosidic linkage between uracil and ribose. In the two-step degradation of pseudouridine, pseudouridine 5’-monophosphate glycosylase (PUMY) is responsible for the second step and catalyses the cleavage of the C-C glycosidic bond in pseudouridine 5’-monophosphate (ΨMP) into uridine and ribose 5’-phosphate, which are recycled via other metabolic pathways. Structural features of Escherichia coli PUMY have been reported, but the details of the substrate specificity of ΨMP were unknown. Here, we present three crystal structures of Arabidopsis thaliana PUMY in different ligation states and a kinetic analysis of ΨMP degradation. The results indicate that Thr149 and Asn308, which are conserved in the PUMY family, are structural determinants for recognizing the nucleobase of ΨMP. The distinct binding modes of ΨMP and ribose 5’-phosphate also suggest that the nucleobase, rather than the phosphate group, of ΨMP dictates the substrate-binding mode. An open-to-close transition of the active site is essential for catalysis, which is mediated by two α-helices, α11 and α12, near the active site. Mutational analysis validates the proposed roles of the active site residues in catalysis. Our structural and functional analyses provide further insight into the enzymatic features of PUMY towards ΨMP.

KEYWORDS:

• Crystal structure
• C-nucleoside
• metalloenzyme
• conformational changes
• substrate fidelity

Acknowledgments

We thank Minsoo Kim for her comments on a PUKI-dependent ΨMP synthesis and Dr. Ah-Reum Lee for providing us with the cDNA library of A. thaliana.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

SR supervised the project; SHK performed crystallographic experiments of holoenzyme and the complex with citrate, and JL determined the crystal structure of the complex with ΨMP/R5P; JL carried out biochemical and kinetic assays; SR and JL analysed the data and wrote the manuscript with contributions from SHK.

Data availability statement

The atomic coordinates and structural factors have been deposited in the Protein Data Bank (http://www.rcsb.org) under ID code 8K05, 8K06, and 8K07.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15476286.2023.2293340

Additional information

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) [2021R1A2C2092118 and RS-2023-00207820].