7K09

Puromycin N-acetyltransferase in complex with acetyl-CoA

7K09 の概要

• エントリーDOI: 10.2210/pdb7k09/pdb
• 分子名称: Puromycin N-acetyltransferase, ACETYL COENZYME *A (3 entities in total)
• 機能のキーワード: enzyme acetyletransferase complex selection marker, antibiotic, transferase
• 由来する生物種: Streptomyces alboniger
• タンパク質・核酸の鎖数: 6
• 化学式量合計: 140346.37

構造登録者

Caputo, A.T.,Newman, J.,Adams, T.E.,Peat, T.S. (登録日: 2020-09-03, 公開日: 2021-03-17, 最終更新日: 2023-10-18)

主引用文献

Caputo, A.T.,Eder, O.M.,Bereznakova, H.,Pothuis, H.,Ardevol, A.,Newman, J.,Nuttall, S.,Peat, T.S.,Adams, T.E.
Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins.
Sci Rep, 11:5247-5247, 2021

PubMed Abstract: Puromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

PubMed: 33664348
DOI: 10.1038/s41598-021-84551-9

実験手法

X-RAY DIFFRACTION (2.313 Å)