7k09: Difference between revisions
No edit summary |
No edit summary |
||
| (One intermediate revision by the same user not shown) | |||
| Line 1: | Line 1: | ||
==Puromycin N-acetyltransferase in complex with acetyl-CoA== | |||
<StructureSection load='7k09' size='340' side='right'caption='[[7k09]], [[Resolution|resolution]] 2.31Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[7k09]] is a 6 chain structure with sequence from [https://en.wikipedia.org/wiki/Streptomyces_alboniger Streptomyces alboniger]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=7K09 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=7K09 FirstGlance]. <br> | |||
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.313Å</td></tr> | |||
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=ACO:ACETYL+COENZYME+*A'>ACO</scene></td></tr> | |||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=7k09 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=7k09 OCA], [https://pdbe.org/7k09 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=7k09 RCSB], [https://www.ebi.ac.uk/pdbsum/7k09 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=7k09 ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[https://www.uniprot.org/uniprot/PUAC_STRAD PUAC_STRAD] Detoxification of puromycin. | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
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. | |||
Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins.,Caputo AT, Eder OM, Bereznakova H, Pothuis H, Ardevol A, Newman J, Nuttall S, Peat TS, Adams TE Sci Rep. 2021 Mar 4;11(1):5247. doi: 10.1038/s41598-021-84551-9. PMID:33664348<ref>PMID:33664348</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
[[Category: | </div> | ||
<div class="pdbe-citations 7k09" style="background-color:#fffaf0;"></div> | |||
== References == | |||
<references/> | |||
__TOC__ | |||
</StructureSection> | |||
[[Category: Large Structures]] | |||
[[Category: Streptomyces alboniger]] | |||
[[Category: Adams TE]] | |||
[[Category: Caputo AT]] | |||
[[Category: Newman J]] | |||
[[Category: Peat TS]] | |||
Latest revision as of 18:15, 18 October 2023
Puromycin N-acetyltransferase in complex with acetyl-CoAPuromycin N-acetyltransferase in complex with acetyl-CoA
Structural highlights
FunctionPUAC_STRAD Detoxification of puromycin. Publication Abstract from PubMedPuromycin 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. Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins.,Caputo AT, Eder OM, Bereznakova H, Pothuis H, Ardevol A, Newman J, Nuttall S, Peat TS, Adams TE Sci Rep. 2021 Mar 4;11(1):5247. doi: 10.1038/s41598-021-84551-9. PMID:33664348[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
|
| ||||||||||||||||||