Characterization of polymers of nucleotide biosynthetic enzymes

Abstract

Mammalian cells contain various proteins that assemble into filamentous structures. Examples include cytoskeletal proteins such as actin and even metabolic enzymes like acetyl-CoA carboxylase. Within the last decade, CTP synthase (CTPS) and inosine monophosphate dehydrogenase (IMPDH) have been found to assemble into filaments. Both are rate-limiting enzymes involved in nucleotide biosynthesis. Their assemblies are linked to nucleotide depletion. Filaments containing CTPS and/or IMPDH have been identified within the cells of different organisms such as bacteria, yeast, flies and mammals. CTPS and IMPDH2 can co-assemble into filaments. To determine whether additional proteins can co-assemble with CTPS and IMPDH2, we implemented the Bio-ID system followed by mass spectrometry. Histidine ammonia lyase was identified as a candidate and preliminary immunofluorescence studies found that it localizes to the filaments. Histidine ammonia lyase releases ammonia in its enzymatic reaction which may be used by CTPS which requires ammonia. CTPS and IMPDH2 are also capable of assembling individually into filaments. CTPS filaments can regulate both cell shape and enzymatic activity within bacterial cells. On the other hand, the function of IMPDH2 polymerization is unclear. What is known is that IMPDH2 polymerization can be regulated by the binding of its allosteric regulators GTP and ATP which bind to the Bateman domain of the enzyme. ATP triggers the assembly of enzymatically active filaments composed of extended octamers. GTP, on the other hand, promotes enzymatically inactive octamers which are compressed in form and it is unknown whether these octamers can polymerize. To determine whether IMPDH2 polymerization changed IMPDH2 enzymatic activity, point mutants of IMPDH2 that either prevent or reinforced octamer polymerization were engineered. No difference in activity, substrate affinity, or GTP inhibition sensitivity was found between polymerized and non-polymerized IMPDH2. Cellular isotope tracing experiments validated these results. Surprisingly, electron microscopy revealed that compact octamers can polymerize. These findings demonstrate that IMPDH 2 polymerization has no effect on enzymatic activity. IMPDH is also involved in activities unrelated to its enzymatic activity such as gene regulation. Polymerization may be a way to regulate its involvement in these activities.%%%%Ph.D., Molecular and Cell Biology and Genetics – Drexel University, 2017