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

Marta Grzechowiak,Mariusz Jaskolski,Milosz Ruszkowski,Joanna Sliwiak

doi:10.3389/fpls.2020.00754

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 D3-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.

Highlights

As a constituent of many important bioorganic compounds, nitrogen is one of the most essential macroelements in the biosphere
To provide background for functional and structural discussions, we investigated the evolutionary divergence of the Glutamate dehydrogenase (GDH) enzymes
We propose to designate the cluster of sequences containing AtGDH1 and AtGDH3 as type I, and the cluster which contains AtGDH2 as type II

Summary

INTRODUCTION

As a constituent of many important bioorganic compounds, nitrogen is one of the most essential macroelements in the biosphere. GS catalyzes the ATP-dependent conversion of glutamate into glutamine by incorporating ammonia, whereas GOGAT transfers the amide group from glutamine to 2-oxoglutarate (2OG), producing two molecules of glutamate These two enzymes function in a cycle in the cytosol and chloroplasts. The GS-GOGAT cycle is recognized as the major route of ammonia assimilation into organic molecules in plants (Lea and Miflin, 1974; Miflin and Lea, 1980). The major role of GDH lies in fueling the tricarboxylic acid cycle (TCA cycle) with 2OG when carbon becomes the limiting factor (Fontaine et al, 2012) This way – together with GOGAT – GDH controls Glu homeostasis (Labboun et al, 2009). The structures, together with phylogenetic, biochemical, and biophysical data, provide a complex overview of the AtGDH1 enzyme

RESULTS AND DISCUSSION

CONCLUSION

MATERIALS AND METHODS

DATA AVAILABILITY STATEMENT