5TSU

Active conformation for Engineered human cystathionine gamma lyase (E59N, R119L, E339V) to depleting methionine

5TSU の概要

• エントリーDOI: 10.2210/pdb5tsu/pdb
• 関連するPDBエントリー: 5TT2
• 分子名称: Cystathionine gamma-lyase, METHIONINE, SULFATE ION, ... (7 entities in total)
• 機能のキーワード: engineered protein, methioninase, inactive form, lyase
• 由来する生物種: Homo sapiens (Human); 詳細
• タンパク質・核酸の鎖数: 8
• 化学式量合計: 373777.56

構造登録者

Yan, W.,Zhang, Y. (登録日: 2016-10-31, 公開日: 2017-10-11, 最終更新日: 2024-12-25)

主引用文献

Yan, W.,Stone, E.,Zhang, Y.J.
Structural Snapshots of an Engineered Cystathionine-gamma-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site.
Biochemistry, 56:876-885, 2017

PubMed Abstract: Enzyme therapeutics that can degrade l-methionine (l-Met) are of great interest as numerous malignancies are exquisitely sensitive to l-Met depletion. To exhaust the pool of methionine in human serum, we previously engineered an l-Met-degrading enzyme based on the human cystathionine-γ-lyase scaffold (hCGL-NLV) to circumvent immunogenicity and stability issues observed in the preclinical application of bacterially derived methionine-γ-lyases. To gain further insights into the structure-activity relationships governing the chemistry of the hCGL-NLV lead molecule, we undertook a biophysical characterization campaign that captured crystal structures (2.2 Å) of hCGL-NLV with distinct reaction intermediates, including internal aldimine, substrate-bound, gem-diamine, and external aldimine forms. Curiously, an alternate form of hCGL-NLV that crystallized under higher-salt conditions revealed a locally unfolded active site, correlating with inhibition of activity as a function of ionic strength. Subsequent mutational and kinetic experiments pinpointed that a salt bridge between the phosphate of the essential cofactor pyridoxal 5'-phosphate (PLP) and residue R62 plays an important role in catalyzing β- and γ-eliminations. Our study suggests that solvent ions such as NaCl disrupt electrostatic interactions between R62 and PLP, decreasing catalytic efficiency.

PubMed: 28106980
DOI: 10.1021/acs.biochem.6b01172

実験手法

X-RAY DIFFRACTION (2.2 Å)