Structurally disordered C-terminal residues of GTP cyclohydrolase II are essential for its enzymatic activity

Abstract

GTP cyclohydrolase II (GCHII) is one of the rate limiting enzymes in riboflavin biosynthesis pathway and is shown to be a potential drug target for most of the pathogens. Previous biochemical and structural studies have identified the active site residues and elucidated the steps involved in the catalytic mechanism of GCHII. However, the last ∼20–25 C-terminal residues of GCHII remains unstructured in all the crystal structures determined to date and their role in the catalytic activity, if any, remains elusive. Therefore, to understand the role of these unstructured C-terminal residues, a series of C-terminal deletion mutants of GCHII from Helicobacter pylori (hGCHII) were generated and their catalytic activity was compared with its wild-type. Surprisingly, none of the C-terminal deletion mutants shows any enzymatic activity indicating that these are essential for GCHII function. To get additional insights for such loss of activity, homology models of full-length and deletion mutants of hGCHII in complex with GTP, Mg²⁺, and Zn²⁺ were generated and subjected to molecular dynamics simulation studies. The simulation studies show that a conserved histidine at 190^th position from the unstructured C-terminal region of hGCHII interacts with α-phosphate of GTP. We propose that His-190 may play a role in the hydrolysis of pyrophosphate from GTP and in releasing the product, DARP. In summary, we demonstrate that the unstructured C-terminal residues of GCHII are important for its enzymatic activity and must be considered during rational drug designing.

Communicated by Ramaswamy H. Sarma

Keywords:

• GTP cyclohydrolase II
• C-terminal disorderliness
• molecular dynamics simulations
• riboflavin biosynthesis pathway
• Helicobacter pylori

Acknowledgments

SY and SS acknowledge Council of Scientific and Industrial Research (CSIR), Government of India for the doctoral fellowship. RN acknowledges CSIR, India for his post-doctoral fellowship. Authors also acknowledges the financial support from Council of Scientific and Industrial Research (CSIR) for the project ‘Emerging and re-emerging challenges in infectious diseases: Systems based drug design for infectious diseases (SPlenDID)’ during time period of 2012–2016.

Disclosure statement

There is no potential conflict of interest among authors.

Additional information

Funding

This work was funded by Council of Scientific and Industrial Research, India.

Notes on contributors

Savita Yadav

SK and SY conceived the idea. SY and SS performed, interpreted, and analyzed the experimental studies. RS performed and analyzed the MD simulation studies. BS supervised the simulation studies. All authors contributed in writing and proof reading of the manuscript.

Suruchi Singh

SK and SY conceived the idea. SY and SS performed, interpreted, and analyzed the experimental studies. RS performed and analyzed the MD simulation studies. BS supervised the simulation studies. All authors contributed in writing and proof reading of the manuscript.

Subramanian Karthikeyan

SK and SY conceived the idea. SY and SS performed, interpreted, and analyzed the experimental studies. RS performed and analyzed the MD simulation studies. BS supervised the simulation studies. All authors contributed in writing and proof reading of the manuscript.