Application of Acetyl-CoA synthetase from Methanothermobacter thermautotrophicus to non-native substrates

The substrate selectivity of the Trp⁴¹⁶Gly mutant of Methanothermobacter thermautotrophicus acetyl-CoA synthetase (Trp⁴¹⁶Gly MT-ACS1) was explored. The goal was to identify its substrate range, particularly for functionalized carboxylic acid substrates that would allow post-synthesis functionalization of CoA thioesters or downstream products using metathesis or Click chemistry. Relative activities were determined by in situ formation of acyl-hydroxamate iron (III) complexes. Trp⁴¹⁶Gly MT-ACS1 showed good activities for saturated straight chain carboxylic acids from C₂ to C₈, for ω-alkenyl straight chain carboxylic acids from C₄ to C₇ and for ω-alkynyl straight chain carboxylic acids from C₅ to C₇. Carboxylic acids showing ≥20% conversion in screening reactions were used in preparative conversions that completely consumed the added CoASH.