Aminoacyl-coenzyme A synthesis catalyzed by adenylation domains.

Adenylate forming enzymes play an important role in nature as they are involved in a number of essential biochemical pathways. In this study, we investigated the ability of a set of structurally related recombinant bacterial adenylate forming enzymes derived from nonribosomal peptide synthetases for their ability to synthesize acyl-CoAs in vitro. Adenylation-domains normally transfer their reactive aminoacyl-adenylates onto the covalently attached 4'-phosphopantetheine moiety of small carrier proteins. In detail, DltA, DhbE, GrsA-A, TycB(3)-A, and TycC(3)-A were investigated for their ability to synthesize acyl-CoAs. As reference, acetyl-CoA-synthetase (Acs) of B. subtilis was utilized, which naturally synthesizes acetyl-CoA from acetate, CoA-SH and ATP. Interestingly, all enzymes were capable of producing acyl-CoAs, albeit with differing efficiencies. Surprisingly, both CoA-SH and ATP were observed to inhibit the adenylation reaction at higher concentrations. Product quantification for kinetic determination was carried out by ESI-SIM-MS. Our results allow speculation as to evolutionary relationships within the large class of adenylate forming enzymes.