The substrate selectivity of the Trp(416)Gly mutant of Methanothermobacter thermautotrophicus acetyl-CoA synthetase (Trp(416)Gly 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. Trp(416)Gly MT-ACS1 showed good activities for saturated straight chain carboxylic acids from C-2 to C-8, for omega-alkenyl straight chain carboxylic acids from C-4 to C-7 and for co-alkynyl straight chain carboxylic acids from C-5 to C-7. Carboxylic acids showing >= 20% conversion in screening reactions were used in preparative conversions that completely consumed the added CoASH.