7K0A
Puromycin N-acetyltransferase in complex with acetylated puromycin and CoA
Summary for 7K0A
| Entry DOI | 10.2210/pdb7k0a/pdb |
| Descriptor | Puromycin N-acetyltransferase, N-Acetylpuromycin, COENZYME A, ... (7 entities in total) |
| Functional Keywords | enzyme acetyletransferase complex selection marker, antibiotic, transferase |
| Biological source | Streptomyces alboniger |
| Total number of polymer chains | 2 |
| Total formula weight | 48699.96 |
| Authors | Peat, T.S.,Caputo, A.T.,Newman, J.,Adams, T.E. (deposition date: 2020-09-03, release date: 2021-03-17, Last modification date: 2023-10-18) |
| Primary citation | 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 Cited by 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: 33664348DOI: 10.1038/s41598-021-84551-9 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (2 Å) |
Structure validation
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