Biology subjects

Burrell, A. L.

Publications and source records attributed to Burrell, A. L..

3 recordsLinked to original sources

bioRxiv2023

Light-sensitive phosphorylation regulates enzyme activity and filament assembly of human IMPDH1 retinal splice variants

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo guanosine triphosphate (GTP) synthesis and is controlled by feedback inhibition and allosteric regulation. IMPDH assembles into micron-scale filaments in cells, which desensitizes the enzyme to feedback inhibition by GTP and boosts nucleotide production. The vertebrate retina expresses two tissue-specific splice variants IMPDH1(546) and IMPDH1(595). IMPDH1(546) filaments adopt high and low activity conformations, while IMPDH1(595) filaments maintain high activity. In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of phosphorylation in IMPDH1 variants. The S477D mutation re-sensitized both variants to GTP inhibition, but only blocked assembly of IMPDH1(595) filaments and not IMPDH1(546) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of the high activity assembly interface, still allowing assembly of low activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, phosphorylation at S477 acts as a mechanism for downregulating retinal GTP synthesis in the dark, when nucleotide turnover is decreased. Like IMPDH1, many other metabolic enzymes dynamically assemble filamentous polymers that allosterically regulate activity. Our work suggests that posttranslational modifications may be yet another layer of regulatory control to finely tune activity by modulating filament assembly in response to changing metabolic demands. SIGNIFICANCE STATEMENTOver 20 different metabolic enzymes form micron-scale filaments in cells, suggesting that filament assembly is a conserved mechanism for regulating diverse metabolic pathways. Filament assembly regulates catalytic activity of many of these enzymes, including inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in de novo GTP biosynthesis. The vertebrate retina expresses two IMPDH1 splice variants that are critical for maintaining nucleotide levels required for phototransduction. Here, we show that filament assembly by these variants is itself controlled by phosphorylation at a single residue, adding further complexity to the tight regulation of nucleotide metabolism in the retina. Phosphorylation and other posttranslational modifications are likely to be a general regulatory mechanism controlling filament assembly by enzymes in many different metabolic pathways.

biochemistry↗

bioRxiv2023

Point mutations in IMPDH2 which cause early-onset neurodevelopmental disorders disrupt enzyme regulation and filament structure

Inosine 5 monophosphate dehydrogenase (IMPDH) is a critical regulatory enzyme in purine nucleotide biosynthesis that is inhibited by the downstream product GTP. Multiple point mutations in the human isoform IMPDH2 have recently been associated with dystonia and other neurodevelopmental disorders, but the effect of the mutations on enzyme function has not been described. Here, we report identification of two additional affected individuals with missense variants in IMPDH2 and show that all of the disease-associated mutations disrupt GTP regulation. Cryo-EM structures of one IMPDH2 mutant suggest this regulatory defect arises from a shift in the conformational equilibrium toward a more active state. This structural and functional analysis provides insight into IMPDH2-associated disease mechanisms that point to potential therapeutic approaches and raises new questions about fundamental aspects of IMPDH regulation. Significance StatementPoint mutations in the human enzyme IMPDH2, a critical regulator of nucleotide biosynthesis, are linked to neurodevelopmental disorders, such as dystonia. Here, we report two additional IMPDH2 point mutants associated with similar disorders. We investigate the effects of each mutation on IMPDH2 structure and function in vitro and find that all mutations are gain of function, preventing allosteric regulation of IMPDH2 activity. We report high resolution structures of one variant and present a structure-based hypothesis for its dysregulation. This work provides a biochemical basis for understanding diseases caused by IMPDH2 mutation and lays a foundation for future therapeutic development.

biochemistry↗

bioRxiv2017

IMPDH polymers accommodate both catalytically active and inactive conformations

Several metabolic enzymes undergo reversible polymerization into macromolecular assemblies. The function of these assemblies is often unclear but in some cases they regulate enzyme activity and metabolic homeostasis. The guanine nucleotide biosynthetic enzyme inosine monophosphate dehydrogenase (IMPDH) forms octamers that polymerize into helical chains. In mammalian cells, IMPDH filaments can associate into micron-length assemblies. Polymerization and enzyme activity are regulated in part by binding of purine nucleotides to an allosteric regulatory domain. ATP promotes octamer polymerization, whereas GTP promotes a compact, inactive conformation whose ability to polymerize is unknown. An open question is whether polymerization directly alters IMPDH catalytic activity. To address this, we identified point mutants of human IMPDH2 that either prevent or promote polymerization. Unexpectedly, we found that polymerized and non-assembled forms of IMPDH have comparable catalytic activity, substrate affinity, and GTP sensitivity and validated this finding in cells. Electron microscopy revealed that substrates and allosteric nucleotides shift the equilibrium between active and inactive conformations in both the octamer and the filament. Unlike other metabolic filaments, which selectively stabilize active or inactive conformations, IMPDH filaments accommodate multiple states. Thus, although polymerization alone does not impact catalytic activity, substrate availability and purine balance dramatically affect IMPDH filament architecture.

biochemistry↗