Crystal structure of the 2‐iminoglutarate‐bound complex of glutamate dehydrogenase from <i>Corynebacterium glutamicum</i>

Tanaka, Takeo; Yin, Lulu; Nakamura, Shugo; Kosono, Saori; Kuzuyama, Tomohisa; Nishiyama, Makoto

doi:10.1002/1873-3468.12667

Cited by 17 publications

(21 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The Lys122 of AnGDH satisfying this ionic interaction is highly conserved in NADP + -dependent GDHs. The similar role for an equivalent lysine residue in CgGDH has also been proposed (30,44). The O3 oxygen atom of the 2'-phosphate may remain neutral or be negatively charged.…”

Section: Nadpsupporting

confidence: 58%

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

Prakash

Punekar

Bhaumik

2018

Journal of Biological Chemistry

View full text Add to dashboard Cite

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...

show abstract

Section: Nadpsupporting

confidence: 58%

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

Prakash

Punekar

Bhaumik

2018

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Tartrate has maximum interactions and forms an additional hydrogen bond interaction with the Asp154, when compared to succinate and malonate. Notably, one subunit of the previously reported hexameric structure (5GUD) of C. glutamicum GDH (CgGDH)42 shows open conformation although it has citrate and NADP + bound in the active site. A structural comparison of CgGDH with AtGDH-TLA-NADPH shows a slightly different binding mode of citrate as compared to tartrate (FigureS3).…”

mentioning

confidence: 99%

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

et al. 2021

View full text Add to dashboard Cite

Glutamate dehydrogenase (GDH) is a salient metabolic enzyme which catalyzes the NAD + -or NADP + -dependent reversible conversion of α-ketoglutarate (AKG) to L-glutamate; and thereby connects the carbon and nitrogen metabolism cycles in all living organisms. The function of GDH is extensively regulated by both metabolites (citrate, succinate, etc.) and non-metabolites (ATP, NADH, etc.) but sufficient molecular evidences are lacking to rationalize the inhibitory effects by the metabolites. We have expressed and purified NADP + -dependent Aspergillus terreus GDH (AtGDH) in recombinant form. Succinate, malonate, maleate, fumarate, and tartrate independently inhibit the activity of AtGDH to different extents. The crystal structures of AtGDH complexed with the dicarboxylic acid metabolites and the coenzyme NADPH have been determined. Although AtGDH structures are not complexed with substrate; surprisingly, they acquire super closed conformation like previously reported for substrate and coenzyme bound catalytically competent Aspergillus niger GDH (AnGDH). These dicarboxylic acid metabolites partially occupy the same binding pocket as substrate; but interact with varying polar interactions and the coenzyme NADPH binds to the Domain-II of AtGDH. The low inhibition potential of tartrate as compared to other dicarboxylic acid metabolites is due to its weaker interactions of carboxylate groups with AtGDH. Our results suggest that the length of carbon skeleton and positioning of the carboxylate groups of inhibitors between two conserved lysine residues at the GDH active site might be the determinants of their inhibitory potency. Molecular details on the dicarboxylic acid metabolites bound AtGDH active site architecture presented here would be applicable to GDHs in general.

show abstract

“…To clarify the reasons for the configuration of this specific product, the docking results and catalytic mechanism of GDH were analyzed. As shown in Figure 7A , two residues (K92 and S380 of the wild-type or Ec GDH K116Q/N348M ) formed hydrogen bones with the γ-carboxyl group of 2-ketoglutarate or the carboxyl group of LA, and the substrate carbonyl group was stabilized by the side chain of K126 before a hydride was supplied by the NADPH to the carbonyl carbon atom ( Tomita et al, 2017 ). Moreover, the coenzyme NADPH attacked the 2-ketoglutarate from the re face ( Figure 7B ).…”

Section: Resultsmentioning

confidence: 99%

A Sustainable Approach for Synthesizing (R)-4-Aminopentanoic Acid From Levulinic Acid Catalyzed by Structure-Guided Tailored Glutamate Dehydrogenase

Zhou

et al. 2022

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

In this study, a novel enzymatic approach to transform levulinic acid (LA), which can be obtained from biomass, into value-added (R)-4-aminopentanoic acid using an engineered glutamate dehydrogenase from Escherichia coli (EcGDH) was developed. Through crystal structure comparison, two residues (K116 and N348), especially residue 116, were identified to affect the substrate specificity of EcGDH. After targeted saturation mutagenesis, the mutant EcGDHK116C, which was active toward LA, was identified. Screening of the two-site combinatorial saturation mutagenesis library with EcGDHK116C as positive control, the kcat/Km of the obtained EcGDHK116Q/N348M for LA and NADPH were 42.0- and 7.9-fold higher, respectively, than that of EcGDHK116C. A molecular docking investigation was conducted to explain the catalytic activity of the mutants and stereoconfiguration of the product. Coupled with formate dehydrogenase, EcGDHK116Q/N348M was found to be able to convert 0.4 M LA by more than 97% in 11 h, generating (R)-4-aminopentanoic acid with >99% enantiomeric excess (ee). This dual-enzyme system used sustainable raw materials to synthesize (R)-4-aminopentanoic acid with high atom utilization as it utilizes cheap ammonia as the amino donor, and the inorganic carbonate is the sole by-product.

show abstract

Crystal structure of the 2‐iminoglutarate‐bound complex of glutamate dehydrogenase from Corynebacterium glutamicum

Abstract: The atomic coordinate and structure factors have been deposited in the RCSB PDB database under the accession number 5GUD.

Cited by 17 publications

References 47 publications

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

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

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

A Sustainable Approach for Synthesizing (R)-4-Aminopentanoic Acid From Levulinic Acid Catalyzed by Structure-Guided Tailored Glutamate Dehydrogenase

Contact Info

Product

Resources

About