Table 2

Substrate requirements for synthesis of acyl-HSLs by RhlI

Substrates addedAcyl-HSL produced, nmol⋅min−1⋅mg−1 RhlI
Butyryl-ACP and SAM650 (530)
Butyryl-ACP, SAM, and NADPH720
Butyryl-CoA, HSL, and NADPH<0.02
Butyryl-CoA and SAM80 (40)
Butyrate and SAM<0.02
Butyryl-SAM(80)
Hexanoyl-ACP and SAM260
Hexanoyl-CoA and SAM<0.02
Octanoyl-ACP and SAM30
Decanoyl-ACP and SAM<0.02
Butyryl-ACP and S-adenosylhomocysteine(<0.5)
Butyryl-ACP and S-adenosylcysteine(<0.5)
Butyryl-ACP and HSL(<0.5)
Butyryl-ACP and homocysteine(1)
Butyryl-ACP and homoserine(<0.5)
Butyryl-ACP and methionine(<0.5)
  • Each reaction mixture contained 72 ng of purified RhlI plus the indicated amino donor at 60 μM and the indicated acyl donor at 40 μM. For butyryl-SAM, the concentration was 200 μM. Where indicated, 500 μM NADPH was included in the reaction mixture. As described in Materials and Methods, the specific activity of RhlI was determined by measuring the production of 14C-labeled acyl-HSL. The values in parenthesis are obtained by using bioassays to measure acyl-HSLs.