The 1.64 A structure of the apoenzyme form of saccharopine dehydrogenase (SDH) from Saccharomyces cerevisiae shows the enzyme to be composed of two domains with similar dinucleotide binding folds with a deep cleft at the interface. The structure reveals homology to alanine dehydrogenase, despite low primary sequence similarity. A model of the ternary complex of SDH, NAD, and saccharopine identifies residues Lys77 and Glu122 as potentially important for substrate binding and/or catalysis, consistent with a proton shuttle mechanism. Furthermore, the model suggests that a conformational change is required for catalysis and that residues Lys99 and Asp281 may be instrumental in mediating this change. Analysis of the crystal structure in the context of other homologous enzymes from pathogenic fungi and human sources sheds light into the suitability of SDH as a target for antimicrobial drug development.
"In the references, some residues located at the active site have been proposed to sever as general acid–base 1 and/or 2, including Lys77, Glu122, Glu78, Asp271 and His96   . However, all of these proposals were given on the basis of structures with the active site in open or partially closed form, or the semiempirical models. "
[Show abstract][Hide abstract] ABSTRACT: Saccharopine dehydrogenase (SDH) is the last enzyme in the AAA pathway of l-lysine biosynthesis. On the basis of crystal structures of SDH, the whole catalytic cycle of SDH has been studied by using density functional theory (DFT) method. Calculation results indicate that hydride transfer is the rate-limiting step with an energy barrier of 25.02kcal/mol, and the overall catalytic reaction is calculated to be endothermic by 9.63kcal/mol. Residue Lys77 is proved to be functional only in the process of saccharopine deprotonation until the formation of product l-lysine, and residue His96 is confirmed to take part in multiple proton transfer processes and can be described as a proton transfer station. From the point of view of energy, the SDH catalytic reaction for the synthesis of l-lysine is unfavorable compared with its reverse reaction for the synthesis of saccharopine. These results are essentially consistent with the experimental observations from pH dependence of kinetic parameters and isotope effects.
[Show abstract][Hide abstract] ABSTRACT: Three structures of saccharopine dehydrogenase (l-lysine-forming) (SDH) have been determined in the presence of sulfate, adenosine monophosphate (AMP), and oxalylglycine (OxGly). In the sulfate-bound structure, a sulfate ion binds in a cleft between the two domains of SDH, occupies one of the substrate carboxylate binding sites, and results in partial closure of the active site of the enzyme due to a domain rotation of almost 12 degrees in comparison to the apoenzyme structure. In the second structure, AMP binds to the active site in an area where the NAD+ cofactor is expected to bind. All of the AMP moieties (adenine ring, ribose, and phosphate) interact with specific residues of the enzyme. In the OxGly-bound structure, carboxylates of OxGly interact with arginine residues representative of the manner in which substrate (alpha-ketoglutarate and saccharopine) may bind. The alpha-keto group of OxGly interacts with Lys77 and His96, which are candidates for acid-base catalysis. Analysis of ligand-enzyme interactions, comparative structural analysis, corroboration with kinetic data, and discussion of a ternary complex model are presented in this study.
[Show abstract][Hide abstract] ABSTRACT: Octopine dehydrogenase [N(2)-(D-1-carboxyethyl)-L-arginine:NAD(+) oxidoreductase] (OcDH) from the adductor muscle of the great scallop Pecten maximus catalyzes the reductive condensation of l-arginine and pyruvate to octopine during escape swimming. This enzyme, which is a prototype of opine dehydrogenases (OpDHs), oxidizes glycolytically born NADH to NAD(+), thus sustaining anaerobic ATP provision during short periods of strenuous muscular activity. In contrast to some other OpDHs, OcDH uses only l-arginine as the amino acid substrate. Here, we report the crystal structures of OcDH in complex with NADH and the binary complexes NADH/l-arginine and NADH/pyruvate, providing detailed information about the principles of substrate recognition, ligand binding and the reaction mechanism. OcDH binds its substrates through a combination of electrostatic forces and size selection, which guarantees that OcDH catalysis proceeds with substrate selectivity and stereoselectivity, giving rise to a second chiral center and exploiting a "molecular ruler" mechanism.
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