Structural basis for the catalytic mechanism and α-ketoglutarate cooperativity of glutamate dehydrogenase

Abstract

Glutamate dehydrogenase (GDH) is a key enzyme connecting carbon and nitrogen metabolism in all living organisms. Despite extensive studies on GDHs from both prokaryotic and eukaryotic organisms in the last 40 years, the structural basis of the catalytic features of this enzyme remains incomplete. This study reports the structural basis of the GDH catalytic mechanism and allosteric behavior. We determined the first high-resolution crystal structures of glutamate dehydrogenase from the fungus Aspergillus niger (AnGDH), a unique NADP + -dependent allosteric enzyme that is forward inhibited by the formation of mixed disulfide. We determined the structures of the active enzyme in its apo form and in binary/ternary complexes with bound substrate (α-ketoglutarate), inhibitor (isophthalate), coenzyme (NADPH), or two reaction intermediates (α-iminoglutarate and 2-amino-2-hydroxyglutarate). The structure of the forward-inhibited enzyme (fiAnGDH) was also determined. The hexameric AnGDH had three open subunits at one side and three partially closed protomers at the other, a configuration not previously been reported. The AnGDH hexamers having subunits with different conformations indicated that its α-ketoglutaratedependent homotropic cooperativity follows the Monod-Wyman-Changeux (MWC) model. Moreover, the position of the water attached to Asp154 and Gly153 defined the previously unresolved ammonium ion-binding pocket, and the binding site for the 2'-phosphate group of the coenzyme was also better defined by our structural data. Additional structural and mutagenesis experiments identified the residues essential for coenzyme recognition. This study reveals the structural features responsible for positioning α-ketoglutarate, NADPH, ammonium ion, and the reaction intermediates in the GDH active site.Enzymes are important biological macromolecules and their catalytic functions govern a number of biological activities in all living organisms. Visualization of the active site of an enzymesubstrate complex or an enzyme bound to the catalytically competent reaction intermediate provides a direct proof of the reaction mechanism (1). Allosteric regulation of enzymes is one of the most fundamental processes that control several cellular activities. Obtaining quantitative molecular description of enzyme allostery has remained a central focus in biology (2). However, trapping the various structural intermediate states to gain detailed understanding about the kinetic properties of allosteric enzymes has remained very challenging (2,3). Glutamate dehydrogenase (GDH) is an oxidoreductase important for ammonia metabolism in archebacteria, eubacteria, and eukaryotes (4,5). We have extensively studied this enzyme to discern the structural basis of unique properties related to its catalytic mechanism and allosteric behavior. GDH catalyzes the reversible oxidation of L-glutamate to α-ketoglutarate and serves as a coupler between carbon and nitrogen metabolism. Structural insights into the catalytic properties of GDH2 the coenzyme spec...

Rational Molecular Engineering of Glutamate Dehydrogenases for Enhancing Asymmetric Reductive Amination of Bulky α‐Keto Acids

“…The three‐dimensional homology model of wild type Pp GluDH was generated with the SWISS‐MODEL server (https://swissmodel.expasy.org/) using the solved structure of GluDH from Corynebacterium glutamicum (PDB ID: 5IJZ, 71.4% sequence identity with Pp GluDH) as a template. According to recently reported catalytic mechanism of GluDH, the whole reductive amination process from α‐KG to L‐glutamate including the formation of imine intermediate is enzyme‐catalyzed. So the α‐KG in Cg GluDH crystal structure was retained in the homology model of Pp GluDH as starting substrate for the analysis of substrate binding mode of Pp GluDH.…”

Temperature-induced reorganisation of Schistocephalus solidus (Cestoda) proteome during the transition to the warm-blooded host

“…Glutamate dehydrogenase has been found directly in trematodes vitellaria (Abidi et al, 2009) and its overexpression was observed in sexually mature parasites S. solidus, Echinococcus granulosus, Opisthorchis felineus and Schistosoma mansoni compared to their larvae and immature adults, indicating a crucial role in flatworm fertility (Protasio et al, 2012;Zheng et al, 2013;Hébert et al, 2017;Ershov et al, 2019). Interestingly, the S. solidus glutamate dehydrogenase isozyme A0A0X3P860 contains the sequence GFGNVG and Glu (278) in the structure, which is more typical for isoforms that provide the deamination reaction rather than the addition of ammonia (Oliveira et al, 2012;Prakash et al, 2018;Grzechowiak et al, 2020).…”

Molecular insights into the inhibition of glutamate dehydrogenase (GDH) by the dicarboxylic acid metabolites

“…Earlier Caughey et al (1957), albeit without structural data, reported that the metabolites with planar conformation and a terminal inter-protonic distance of ~7.65 Å can act as good inhibitors for GDH. 28 The known potent inhibitors of GDH such as isophthalate, 2-methyleneglutarate, oxalylglycine and glutarate might orient and stretch in a way to generate a 5-carbon molecule representation 9,28,36 when they get bound in the enzyme active site. The recent crystal structures of AnGDH reveal that the carboxylate groups of -ketoglutarate and isophthalate occupy the same positions with separation of ~6.4 Å (Figure 6A).…”