6.2.1.14: 6-carboxyhexanoate-CoA ligase

site-directed mutagenesis, the mutant variant displays only a modest 20% loss in activity relative to the wild-type, reflecting the importance of these other interacting residues in stabilizing CoA binding

R159A

site-directed mutagenesis, the activity to hydrolyze adenylates of noncognate substrates is abolished in the mutant. The R159A variant can no longer proofread, but the enzyme still retains ligase activity and can catalyze the formation of pimeloyl-CoA, the mutant demonstrates a notable reduction in turnover, which is in line with the function of the residue in forming the exterior wall of the pimelate-binding cavity

R201A

site-directed mutagenesis, the mutation has little effect on product formation

R215A

site-directed mutagenesis, the mutant demonstrates a substantial reduction in product formation

S182A

site-directed mutagenesis, the mutant demonstrates a substantial reduction in product formation

Y187A

site-directed mutagenesis, the mutant demonstrates a notable reduction in turnover, which is in line with the function of the residue in forming the exterior wall of the pimelate-binding cavity

Y199A

site-directed mutagenesis, the mutation has little effect on product formation

R213A

site-directed mutagenesis, almost inactive mutant

R227E

site-directed mutagenesis, the mutant shows a turnover with the natural substrate pimelic acid that is reduced by around 25fold to about 4% activity remaining compared to the wild-type

R227K

site-directed mutagenesis, the mutant shows a turnover with the natural substrate pimelic acid that is reduced by around 25fold to about 4% activity remaining compared to the wild-type

Y199F

site-directed mutagenesis, the mutant retains 55% activity compared to wild-type, the Y199F mutant is inactive with heptanoic acid and octanoic acid, the Y199F mutant displayed a twofold greater activity with pimelic acid but no improvement with azelaic acid compared to wild-type