Structural Studies of Glutamate Dehydrogenase (Isoform 1) From Arabidopsis thaliana, an Important Enzyme at the Branch-Point Between Carbon and Nitrogen Metabolism

Abstract

Glutamate dehydrogenase (GDH) releases ammonia in a reversible NAD(P) +-dependent oxidative deamination of glutamate that yields 2-oxoglutarate (2OG). In current perception, GDH contributes to Glu homeostasis and plays a significant role at the junction of carbon and nitrogen assimilation pathways. GDHs are members of a superfamily of ELFV (Glu/Leu/Phe/Val) amino acid dehydrogenases and are subdivided into three subclasses, based on coenzyme specificity: NAD +-specific, NAD + /NADP + dual-specific, and NADP +-specific. We determined in this work that the mitochondrial AtGDH1 isozyme from A. thaliana is NAD +-specific. Altogether, A. thaliana expresses three GDH isozymes (AtGDH1-3) targeted to mitochondria, of which AtGDH2 has an extra EF-hand motif and is stimulated by calcium. Our enzymatic assays of AtGDH1 established that its sensitivity to calcium is negligible. In vivo the AtGDH1-3 enzymes form homo-and heterohexamers of varied composition. We solved the crystal structure of recombinant AtGDH1 in the apo-form and in complex with NAD + at 2.59 and 2.03 Å resolution, respectively. We demonstrate also that both in the apo form and in 1:1 complex with NAD + , it forms D 3-symmetric homohexamers. A subunit of AtGDH1 consists of domain I, which is involved in hexamer formation and substrate binding, and of domain II which binds coenzyme. Most of the subunits in our crystal structures, including those in NAD + complex, are in open conformation, with domain II forming a large (albeit variable) angle with domain I. One of the subunits of the AtGDH1-NAD + hexamer contains a serendipitous 2OG molecule in the active site, causing a dramatic (∼25 •) closure of the domains. We provide convincing evidence that the N-terminal peptide preceding domain I is a mitochondrial targeting signal, with a predicted cleavage site for mitochondrial processing peptidase (MPP) at Leu17-Leu18 that is followed by an unexpected potassium coordination site (Ser27, Ile30). We also identified several MPD [(+/-)-2-methyl-2,4-pentanediol] binding sites with conserved sequence. Although AtGDH1 is insensitive to MPD in our assays, the observation of druggable sites opens a potential for non-competitive herbicide design.

S-adenosyl-l-homocysteine Hydrolase: A Structural Perspective on the Enzyme with Two Rossmann-Fold Domains

“…All fragments of the cofactor molecule, including pyrophosphate group, nicotinamide, adenine, and d-ribose moieties are involved in polar and non-polar interactions with the macromolecular environment. It is of note, that similar modes of interactions are observed in other NAD(H)-dependent enzymes [1,6,[39][40][41], however, the cofactor binding is much weaker in those enzymes in comparison to SAHases. A closer inspection of numerous NAD(H)-containing Rossmann-fold enzymes indicates that in SAHases, access of the cofactor molecule to the solvent region is significantly limited (Figure 8a,b).…”

Network Topological Analysis for the Identification of Novel Hubs in Plant Nutrition

“…CYSC1 is co-expressed, under Mo starvation, with 16 proteins whereas it is co-expressed with 11 proteins, under Mo sufficiency ( Table 1 ); interestingly, two glutamate dehydrogenase isoforms, GDH1 and GDH2, are co-expressed with CYSC1, under Mo starvation and Mo sufficiency, respectively, but not under Fe sufficiency or Fe starvation ( Supplementary Figure 5 ). GDHs are enzymes at the branch point between carbon and nitrogen metabolism; both isoforms are localized in mitochondria and GDH2 unless GDH1 is calcium-stimulated ( Grzechowiak et al, 2020 ).…”

Targeting Nitrogen Metabolism and Transport Processes to Improve Plant Nitrogen Use Efficiency

“…N remobilization and reassimilation occurs especially during leaf senescence and mainly involves the activity of the cytosolic GS1, which has been shown to affect NUE (see above; Masclaux-Daubresse et al, 2010 ; Avila-Ospina et al, 2014 ; Havé et al, 2017 ). Glutamate dehydrogenase (GDH) may also have some role in N assimilation (Lam et al, 1996 ; Melo-Oliveira et al, 1996 ; Good et al, 2004 ; Lea and Miflin, 2011 ), but it seems to predominantly function in glutamate deamination and ammonium supply for GS1 (Havé et al, 2017 ; Moison et al, 2018 ) during N remobilization or when C is limited (Robinson et al, 1991 ; Masclaux-Daubresse et al, 2006 ; Fontaine et al, 2012 ; Grzechowiak et al, 2020 ). Efforts to enhance plant performance and NUE by overexpressing GDH have been inconclusive.…”