Rivero-Gamez, A. | bioRxiv Science

bioRxiv2026

Structural Mechanism of Filamentation Induced Dampening of GTP Inhibition of Glutamate Dehydrogenase

Glutamate dehydrogenase (GDH) is a highly regulated key enzyme that catalyzes the reversible oxidative deamination of glutamate to alpha-ketoglutarate, positioning it at a critical hub linking amino acid catabolism to energy production while supplying ammonia for urea synthesis and other nitrogen pathways. Early investigations have shown that bovine GDH (bGDH), which shares 98% sequence identity with its human homolog, assembles into polymeric filaments with altered allosteric responses. Filamentation has only relatively recently been appreciated as a widespread mechanism of enzyme regulation, prompting a reevaluation of these early observations in GDH. Here, we use high resolution cryogenic electron microscopy (cryo EM) to show that bGDH hexamers assemble via reciprocal antenna interactions that oppose the conformational changes associated with GTP inhibition, revealing how filamentation reshapes GDH allostery and with implications for the treatment of human disease.

biochemistry↗

bioRxiv2024

Two-Metal Ion Mechanism of DNA Cleavage by Activated, Filamentous SgrAI

Enzymes that form filamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms polymeric filaments. SgrAI filamentation increases enzymatic activity by up to three orders of magnitude and additionally expands its DNA sequence specificity. Prior studies have suggested a mechanistic model linking the structural changes accompanying SgrAI filamentation to its accelerated DNA cleavage activity. In this model, the conformational changes that are specific to filamentous SgrAI maximize contacts between different copies of the enzyme within the filament and create a second divalent cation binding site in each subunit, which in turn facilitates the DNA cleavage reaction. However, our understanding of the atomic mechanism of catalysis is incomplete. Herein, we present two new structures of filamentous SgrAI solved using cryo-electron microscopy (cryo-EM). The first structure, resolved to 3.3 [A], is of filamentous SgrAI containing an active site mutation that is designed to stall the DNA cleavage reaction, which reveals the enzymatic configuration prior to DNA cleavage. The second structure, resolved to 3.1 [A], is of WT filamentous SgrAI containing cleaved substrate DNA, which reveals the enzymatic configuration at the end of the enzymatic cleavage reaction. Both structures contain the phosphate moiety at the cleavage site and the biologically relevant divalent cation cofactor Mg2+ and define how the Mg2+ cation reconfigures during enzymatic catalysis. The data support a model for the activation mechanism that involves binding of a second Mg2+ in the SgrAI active site as a direct result of filamentation induced conformational changes.

biochemistry↗