Arg40 is critical for stability and activity of Adenylosuccinate lyase; a purine salvage enzyme from Leishmania donovani

• Published in: Archives of biochemistry and biophysics 2024-07, Vol.757, p.110040, Article 110040
• Main Authors: Mochi, Jigneshkumar A., Jani, Jaykumar, Tak, Kiran, Lodhi, Krishna Kumar, Pananghat, Gayathri, Pappachan, Anju
• Format: Article
• Language: English
• Subjects: Adenylosuccinate lyase; Adenylosuccinate Lyase - chemistry; Adenylosuccinate Lyase - genetics; Adenylosuccinate Lyase - metabolism; Arginine - chemistry; Arginine - metabolism; Catalytic Domain; Drug target; Enzyme Stability; Fluorescence spectroscopy; Leishmania; Leishmania donovani - enzymology; Leishmania donovani - genetics; Molecular Dynamics Simulation; Molecular dynamics simulations; Mutagenesis, Site-Directed; Protozoan Proteins - chemistry; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; Purine salvage pathway; Purines - chemistry; Purines - metabolism
• ISSN: 0003-9861; 1096-0384; 1096-0384
• DOI: 10.1016/j.abb.2024.110040

Purine salvage enzymes have been of significant interest in anti-Leishmanial drug development due to the parasite's critical dependence on this pathway for the supply of nucleotides in the absence of a de novo purine synthesis pathway. Adenylosuccinate lyase (ADSL) one of the key enzymes in this pathway is a homo-tetramer, where the active site is formed by residues from three distinct subunits. Analysis of the subunit interfaces of LdADSL, revealed a conserved Arg40 forming critical inter-subunit interactions and also involved in substrate binding. We hypothesized that mutating this residue can affect both the structural stability and activity of the enzyme. In our study, we used biochemical, biophysical, and computational simulation approaches to understand the structural and functional role of Arg40 in LdADSL. We have replaced Arg40 with an Ala and Glu using site directed mutagenesis. The mutant enzymes were similar to wild-type enzyme in secondary structure and subunit association. Thermal shift assays indicated that the mutations affected the protein stability. Both mutants showed decreased specific activities in both forward and reverse directions with significantly weakened affinities towards succinyl-adenosine monophosphate (SAMP). The mutations resulted in changes in C3 loop conformation and D3 domain rotation. Consequently, the orientation of the active site amino acid residues changed resulting in compromised activity and stability. Studies so far have majorly focused on the ADSL active site for designing drugs against it. Our work indicates that an alternative inhibitory mechanism for the enzyme can be designed by targeting the inter-subunit interface. [Display omitted] •Arg40 is located at dimeric interface of LdADSL and is also part of active site of enzyme.•LdADSL-R40A and LdADSL-R40E mutants showed reduced affinity towards the substrate.•The mutants retained the tetrameric structure but showed reduced stability.•Mutations resulted in changed orientation of active site residues and the substrate.•Arg40 is necessary for the stability and catalytic activity of LdADSL.