Mycobacterial inosine-5′-monophosphate dehydrogenase (IMPDH) is a key enzyme involved in purine metabolism, catalysing the conversion of inosine-5′-monophosphate (IMP) to xanthosine-5′-monophosphate (XMP), a rate-limiting step in guanine nucleotide biosynthesis. Because of its essential role in nucleotide synthesis, IMPDH has been studied as a potential target for antimicrobial drug development.
Mycobacterial IMPDH exhibits cooperative enzyme kinetics characterised by allosteric interactions between protomers within the tetrameric and/or octameric IMPDH assembly. However, the molecular mechanism of these inter-chain interactions is poorly understood. The C-terminus plays a crucial role in enzyme activity, forming direct interactions with the active site of neighbouring protomers and, as such, may play a role in the observed cooperativity. Previous structural studies have been limited by the absence of the C-terminus in many resolved structures without bound substrates.
In this study, we aimed to elucidate the molecular basis of IMPDH cooperativity by investigating the role of the C-terminus. Structural analysis of mycobacterial IMPDH using cryo-EM revealed barrel-like densities within the core of the octameric assembly. Computational modelling using AlphaFold2 generated structural models of C-terminal peptides, revealing an unexpected antiparallel beta sheet barrel-like assembly that correlates well with the observed density features. Experimental validation by site-directed mutagenesis targeting specific C-terminal residues resulted in changes in the kinetic parameters of the enzyme, providing crucial mechanistic insights into IMPDH cooperativity.
Our findings not only contribute to a deeper understanding of IMPDH function-structure relationships but also have potential implications for the development of IMPDH-targeting inhibitors.
The project National Institute of virology and bacteriology (Programme EXCELES, ID Project No. LX22NPO5103) - Funded by the European Union - Next Generation EU
We acknowledge CF CF CryoEM of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2018127) and European Regional Development Fund-Project „UP CIISB“ (No.CZ.02.1.01/0.0/0.0/18_046/0015974)