3G7K

Crystal Structure of Methylitaconate-delta-isomerase

3G7K の概要

• エントリーDOI: 10.2210/pdb3g7k/pdb
• 関連するPDBエントリー: 1W61 1XUA 2GKJ 2PW0 2PWZ
• 分子名称: 3-methylitaconate isomerase (2 entities in total)
• 機能のキーワード: dapf family fold, closed conformation, open conformation, isomerase
• 由来する生物種: Eubacterium barkeri (Clostridium barkeri)
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 166288.89

構造登録者

Messerschmidt, A.,Macieira, S.,Velarde, M. (登録日: 2009-02-10, 公開日: 2009-07-21, 最終更新日: 2025-05-28)

主引用文献

Velarde, M.,Macieira, S.,Hilberg, M.,Broker, G.,Tu, S.-M.,Golding, B.T.,Pierik, A.J.,Buckel, W.,Messerschmidt, A.
Crystal structure and putative mechanism of 3-methylitaconate-delta-isomerase from Eubacterium barkeri
J.Mol.Biol., 391:609-620, 2009

PubMed Abstract: 3-Methylitaconate-Delta-isomerase (Mii) participates in the nicotinate fermentation pathway of the anaerobic soil bacterium Eubacterium barkeri (order Clostridiales) by catalyzing the reversible conversion of (R)-3-methylitaconate (2-methylene-3-methylsuccinate) to 2,3-dimethylmaleate. The enzyme is also able to catalyze the isomerization of itaconate (methylenesuccinate) to citraconate (methylmaleate) with ca 10-fold higher K(m) but > 1000-fold lower k(cat). The gene mii from E. barkeri was cloned and expressed in Escherichia coli. The protein produced with a C-terminal Strep-tag exhibited the same specific activity as the wild-type enzyme. The crystal structure of Mii from E. barkeri has been solved at a resolution of 2.70 A. The asymmetric unit of the P2(1)2(1)2(1) unit cell with parameters a = 53.1 A, b = 142.3 A, and c = 228.4 A contains four molecules of Mii. The enzyme belongs to a group of isomerases with a common structural feature, the so-called diaminopimelate epimerase fold. The monomer of 380 amino acid residues has two topologically similar domains exhibiting an alpha/beta-fold. The active site is situated in a cleft between these domains. The four Mii molecules are arranged as a tetramer with 222 symmetry for the N-terminal domains. The C-terminal domains have different relative positions with respect to the N-terminal domains resulting in a closed conformation for molecule A and two distinct open conformations for molecules B and D. The C-terminal domain of molecule C is disordered. The Mii active site contains the putative catalytic residues Lys62 and Cys96, for which mechanistic roles are proposed based on a docking experiment of the Mii substrate complex. The active sites of Mii and the closely related PrpF, most likely a methylaconitate Delta-isomerase, have been compared. The overall architecture including the active-site Lys62, Cys96, His300, and Ser17 (Mii numbering) is similar. This positioning of (R)-3-methylitaconate allows Cys96 (as thiolate) to deprotonate C-3 and (as thiol) to donate a proton to the methylene carbon atom of the resulting allylic carbanion. Interestingly, the active site of isopentenyl diphosphate isomerase type I also contains a cysteine that cooperates with glutamate rather than lysine. It has been proposed that the initial step in this enzyme is a protonation generating a tertiary carbocation intermediate.

PubMed: 19559030
DOI: 10.1016/j.jmb.2009.06.052

実験手法

X-RAY DIFFRACTION (2.7 Å)