Premium
Mapping Key Active Site Residues of the PA3944 Gcn5‐related N‐acetyltransferase
Author(s) -
Variot Cillian,
Baumgartner Jackson,
Houseman Charles,
Zhang Brian,
Joe Layton,
Kuhn Misty L.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.06320
Subject(s) - acetyltransferase , acetyltransferases , active site , polymyxin , biochemistry , arylamine n acetyltransferase , enzyme , mutant , substrate (aquarium) , chemistry , biology , antibiotics , acetylation , ecology , gene
PA3944 is an acetyltransferase from Pseudomonas aeruginosa that acetylates polymyxin antibiotics in vitro. It belongs to the large Gcn5‐related N‐ acetyltransferase (GNAT) superfamily. We previously determined the 3D structure of this enzyme, but were unable to obtain a complex in the presence of polymyxin. Since it is not know which residues in the active of this protein are critical for function and recognition of the polymyxin substrate, we created point mutations of residues in the active site and assayed their kinetic activity. We found two key residues that were critical for activity of the enyzme: one was important for the catalytic mechanism and the other played a structural role. Additionally, we found two mutants that improved the enzymatic activity, which could be used to further explore the possibility of using this enzyme for biotechnological applications to modify polymyxin antibiotics with reduced toxicity. Moreover, these results provide insight into GNAT evolution and how key residues can alter substrate specificity and structure. Support or Funding Information Research reported in this work was supported by the National Science Foundation under Grant Number CHE‐1708863 (to MLK).