Structural and biochemical studies of the large glutamate dehydrogenase from Mycobaterium smegmatis

CHAMORRO, NICOLÁS; LISA, MARÍA-NATALIA

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica; 2022

Reunión Anual de la Sociedad Argentina de Biofísica

Glutamate dehydrogenases (GDHs) are oligomeric enzymes that catalyze the oxidative deamination of L-glutamate in most living beings. They are classified into two subfamilies: small GDHs (S-GDHs) are hexameric, with monomers of 60 kDa, while large GDHs (L-GDHs) are tetrameric, with subunits of 115 or 180 kDa. S-GDHs have been the subject of diverse biochemical and structural studies while L-GDHs have been less studied.The first structural model of a bacterial L-GDH180 was recently obtained by our group by X-ray protein crystallography and cryoEM (Lázaro et al, 2021, Commun. Biol.). The mycobacterial L-GDH180 (mL-GDH180) consists of monomers that contrast with those of S-GDHs by containing long N- and C-terminal extensions flanking the catalytic domain. Such regions are modular and provide the surfaces for oligomerization. Based on features revealed by the 3D structure, it is possible to speculate about their possible role(s) in metabolic sensing. Our goal is to decipher the molecular basis of the activity and the regulation of mL-GDH180 and how it differs from that of S-GDHs.A detailed structural comparison of the catalytic domain of mL-GDH180 from M. smegmatis with that of diverse S-GDHs revealed that secondary structure motifs involved in the oligomerization of S-GDHs are absent in mL-GDH180, explaining the fact that the catalytic domain of mL-GDH180 is not necessary for the oligomerization of this enzyme. Moreover, several insertions were detected in mL-GDH180 as compared to S-GDHs, which could therefore constitute additional sites for allosteric regulation beyond the N- and C-terminal extensions.To analyze the quaternary structure of mL-GDH180 in solution, the enzyme was produced recombinantly and a size-exclusion chromatography was carried out as the last purification step using buffers at different pHs. At pH 7.5 the protein eluted as a mixture of dimeric and tetrameric species while at pH 6.0 it behaved mainly as a tetramer. These observations suggested a modulation of the quaternary structure as a function of pH and is consistent with previous data obtained by single particle cryoEM.Finally, we performed in vitro activity measurements with recombinant mL-GDH180. We estimated, at neutral pH, a Michaelis constant of ca. 10 mM for glutamate, an order of magnitude lower than previously reported by other authors. We will discuss how these results bring us closer to a better understanding of the structural basis of the activity in the subfamily of L-GDHs.