8BPL

Aspartate transcarbamoylase mutant (N2045C, R2238C) from Chaetomium thermophilum CAD-like bound to carbamoyl phosphate

8BPL の概要

• エントリーDOI: 10.2210/pdb8bpl/pdb
• 分子名称: Carbamoyl-phosphate synthase (glutamine-hydrolyzing), PHOSPHORIC ACID MONO(FORMAMIDE)ESTER, GLYCEROL, ... (5 entities in total)
• 機能のキーワード: nucleotide metabolism, de novo pyrimidine synthesis, cad, cysteine, disulfide bridge, protein stability, crosslinking, succinate, multienzymatic protein, transferase
• 由来する生物種: Thermochaetoides thermophila
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 36393.78

構造登録者

del Cano-Ochoa, F.,Ramon-Maiques, S. (登録日: 2022-11-16, 公開日: 2023-02-01, 最終更新日: 2024-10-09)

主引用文献

Del Cano-Ochoa, F.,Rubio-Del-Campo, A.,Ramon-Maiques, S.
A Tailored Strategy to Crosslink the Aspartate Transcarbamoylase Domain of the Multienzymatic Protein CAD.
Molecules, 28:-, 2023

PubMed Abstract: CAD is a 1.5 MDa hexameric protein with four enzymatic domains responsible for initiating de novo biosynthesis of pyrimidines nucleotides: glutaminase, carbamoyl phosphate synthetase, aspartate transcarbamoylase (ATC), and dihydroorotase. Despite its central metabolic role and implication in cancer and other diseases, our understanding of CAD is poor, and structural characterization has been frustrated by its large size and sensitivity to proteolytic cleavage. Recently, we succeeded in isolating intact CAD-like particles from the fungus with high yield and purity, but their study by cryo-electron microscopy is hampered by the dissociation of the complex during sample grid preparation. Here we devised a specific crosslinking strategy to enhance the stability of this mega-enzyme. Based on the structure of the isolated ATC domain, we inserted by site-directed mutagenesis two cysteines at specific locations that favored the formation of disulfide bridges and covalent oligomers. We further proved that this covalent linkage increases the stability of the ATC domain without damaging the structure or enzymatic activity. Thus, we propose that this cysteine crosslinking is a suitable strategy to strengthen the contacts between subunits in the CAD particle and facilitate its structural characterization.

PubMed: 36677714
DOI: 10.3390/molecules28020660

実験手法

X-RAY DIFFRACTION (1.58 Å)