A positive selection approach identifies residues important for folding of Salmonella enterica Pat, an N(ε)-lysine acetyltransferase that regulates central metabolism enzymes.
In Salmonella enterica, the protein acetyltransferase (Pat) enzyme is part of the sirtuin-dependent acylation/deacylation system (SDPADS) that modulates the activity of several proteins via the acylation of lysine residues critical to their activities. Pat is a ~98 kDa protein with two distinct domains, an N-terminal acyl-CoA synthetase (NDP-forming) domain (~700 aa) and a C-terminal acetyltransferase domain (~160 aa), with homology to proteins of the Gcn5-related N-acetyltransferase (GNAT) superfamily. Although the role of the GNAT-like domain is likely responsible for the catalytic activity of Pat, the role of the N-terminal domain remains unclear. Here we report the use of positive selection for identification of residues critical for Pat enzyme activity. This approach revealed seven residues that, when changed, resulted in drastic loss of Pat activity in vitro which caused a discernable loss-of-function phenotype. Five of the seven residues were located in the N-terminal region of Pat and two were located in the GNAT-like domain. Each single-amino-acid variant had a circular dichroism spectrum that differed from that of the wild-type Pat protein, suggesting that loss of enzymatic activity in the mutant proteins was likely due to an inability to acquire its biologically active fold.
Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA. sthaovue@gmail.com
Sandy
S
Thao
Jorge C
JC
Escalante-Semerena
D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism*; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D011487:Protein Conformation; D017510:Protein Folding; D019779:Salmonella enterica / Q000201:enzymology* / Q000235:genetics; D012641:Selection, Genetic
D050505:Mutant Proteins; D000123:Acetyltransferases; C047195:N-epsilon-hydroxylysine acetyltransferase