What is it about?
Shikimate dehydrogenase (EC 1.1.1.25) catalyses the fourth step of the shikimate pathway which is required for the synthesis of the aromatic amino acids and other aromatic compounds in bacteria, microbial eukaryotes, and plants. The crystal structures of the shikimate dehydro- genase AroE from Thermus thermophilus HB8 in its ligand-free form, binary complexes with cofactor NADP+ or substrate shikimate, and the ternary complex with both NADP(H) and shikimate were determined by X-ray diffraction method at atomic resolutions. The crystals are nearly isomor- phous with the asymmetric unit containing a dimer, each subunit of which has a bi-domain structure of compact α/β sandwich folds. The two subunits of the enzyme display asymmetry in the crystals due to different relative orientations between the N- and C-terminal domains resulting in a slightly different closure of the interdomain clefts. NADP(H) is bound to the more closed form only. This closed conformation with apparent higher affinity to the cofactor is also observed in the unliganded crystal form, indicating that the NADP(H) binding to TtAroE may follow the selection mode where the cofactor binds to the subunit that happens to be in the closed conformation in solution. Crystal structures of the closed subunits with and without NADP(H) show no significant structural difference, suggesting that the cofactor binding to the closed subunit corresponds to the lock-and-key model in TtAroE. On the other hand, shikimate binds to both open and closed subunit conformers of both apo and NADP(H)-liganded holo enzyme forms. The ternary complex TtAroE:NADP(H):shikimate allows unambiguous visualization of the SDH permitting elucidation of the roles of conserved residues Lys64 and Asp100 in the hydride ion transfer between NADP(H) and shikimate.
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Why is it important?
To gain more insight into the mechan- isms of substrate/coenzyme binding and catalysis of SDH, we have determined several crystal structures of TtAroE, including its binary com- plexes with cofactor NADP+ (TtAroE:NADP+) or the substrate shikimate (TtAroE:shikimate), and the ternary complex with both NADP(H) and shiki- mate (TtAroE:NADP(H):shikimate). To the best of our knowledge, TtAroE:NADP(H):shikimate com- plex is the first example of the reaction snapshot that allows us to discuss its implication for the SDH reaction mechanism. The observed structural details of shikimate binding may provide an important step in the structure-based design of SDH inhibitors.
Perspectives
We determined crystal structures of TtAroE in a ligand-free form as well as binary and ternary complex forms with the cofactor and the substrate. These structures define the active site of the enzyme and the role of key amino acid residues and provide snapshots of the catalytic cycle. Espe- cially, the first structure of the ternary complex TtAroE:NADP(H):shikimate allowed us to suggest a plausible model for the SDH catalyzed reaction. In this model, Lys64 acts as the base in the initial protonabstraction and Asp100 facilitates the hydride ion transfer between shikimate and C4 of NADP+. Knowledge of the binding modes of substrate and cofactor to this agriculturally and medically impor- tant enzyme will undoubtedly aid in the design of useful inhibitors as new anti-bacterial drugs.
Dr Bagautdin Bagautdinov
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This page is a summary of: Crystal Structures of Shikimate Dehydrogenase AroE from Thermus thermophilus HB8 and its Cofactor and Substrate Complexes: Insights into the Enzymatic Mechanism, Journal of Molecular Biology, October 2007, Elsevier,
DOI: 10.1016/j.jmb.2007.08.017.
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