7LK0

Ornithine Aminotransferase (OAT) cocrystallized with its potent inhibitor - (S)-3-amino-4,4-difluorocyclopent-1-enecarboxylic acid (SS-1-148)

7LK0 の概要

• エントリーDOI: 10.2210/pdb7lk0/pdb
• 分子名称: Ornithine aminotransferase, mitochondrial, (1R,3S)-3-[(E)-({3-hydroxy-2-methyl-5-[(phosphonooxy)methyl]pyridin-4-yl}methylidene)amino]-4-oxocyclopentane-1-carboxylic acid (3 entities in total)
• 機能のキーワード: ornithine aminotransferase, oat, aminotransferase, inhibitor, inactivator, ss-1-148, transferase
• 由来する生物種: Homo sapiens (Human)
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 135697.76

構造登録者

Butrin, A.,Shen, S.,Liu, D.,Silverman, R. (登録日: 2021-02-01, 公開日: 2022-02-16, 最終更新日: 2023-10-18)

主引用文献

Shen, S.,Butrin, A.,Doubleday, P.F.,Melani, R.D.,Beaupre, B.A.,Tavares, M.T.,Ferreira, G.M.,Kelleher, N.L.,Moran, G.R.,Liu, D.,Silverman, R.B.
Turnover and Inactivation Mechanisms for ( S )-3-Amino-4,4-difluorocyclopent-1-enecarboxylic Acid, a Selective Mechanism-Based Inactivator of Human Ornithine Aminotransferase.
J.Am.Chem.Soc., 143:8689-8703, 2021

PubMed Abstract: The inhibition of human ornithine δ-aminotransferase (OAT) is a potential therapeutic approach to treat hepatocellular carcinoma. In this work, ()-3-amino-4,4-difluorocyclopent-1-enecarboxylic acid (SS-1-148, ) was identified as a potent mechanism-based inactivator of OAT while showing excellent selectivity over other related aminotransferases (e.g., GABA-AT). An integrated mechanistic study was performed to investigate the turnover and inactivation mechanisms of . A monofluorinated ketone () was identified as the primary metabolite of in OAT. By soaking OAT holoenzyme crystals with , a precursor to was successfully captured. This -diamine intermediate, covalently bound to Lys292, observed for the first time in OAT/ligand crystals, validates the turnover mechanism proposed for . Co-crystallization yielded OAT in complex with and revealed a novel noncovalent inactivation mechanism in OAT. Native protein mass spectrometry was utilized for the first time in a study of an aminotransferase inactivator to validate the noncovalent interactions between the ligand and the enzyme; a covalently bonded complex was also identified as a minor form observed in the denaturing intact protein mass spectrum. Spectral and stopped-flow kinetic experiments supported a lysine-assisted E2 fluoride ion elimination, which has never been observed experimentally in other studies of related aminotransferase inactivators. This elimination generated the second external aldimine directly from the initial external aldimine, rather than the typical E1cB elimination mechanism, forming a quinonoid transient state between the two external aldimines. The use of native protein mass spectrometry, X-ray crystallography employing both soaking and co-crystallization methods, and stopped-flow kinetics allowed for the detailed elucidation of unusual turnover and inactivation pathways.

PubMed: 34097381
DOI: 10.1021/jacs.1c02456

実験手法

X-RAY DIFFRACTION (1.96 Å)