title: Aminoacyl-coenzyme A synthesis catalyzed by adenylation domains.
abstract: 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.
authors.affiliation: Philipps Universität Marburg, Fachbereich Chemie/Biochemie, Hans-Meerwein-Strasse, 35032 Marburg, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Linne
authors.forename: Antje
authors.initials: A
authors.lastname: Schäfer
authors.forename: Milton T
authors.initials: MT
authors.lastname: Stubbs
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Marahiel
mesh_terms: D000214:Acyl Coenzyme A / Q000096:biosynthesis* / Q000737:chemistry; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019729:Carbon-Oxygen Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019143:Evolution, Molecular; D007700:Kinetics; D008958:Models, Molecular; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D019729:Carbon-Oxygen Ligases; C502532:DltA protein, Bacillus subtilis; D003066:Coenzyme A Ligases; D010453:Peptide Synthases; C035180:tyrocidine synthetase