An Unusual Fatty Acyl:Adenylate Ligase (FAAL)–Acyl Carrier Protein (ACP) Didomain in Ambruticin Biosynthesis

Abstract

The divinylcyclopropane (DVC) fragment of the ambruticins is proposed to be formed by a unique polyene cyclisation mechanism, in which the unusual didomain AmbG plays a key role. It is proposed to activate the branched thioester carboxylic acid resulting from polyene cyclisation and to transfer it to its associated acyl carrier protein (ACP). After oxidative decarboxylation, the intermediate is channelled back into polyketide synthase (PKS) processing. AmbG was previously annotated as an adenylation-thiolation didomain with a very unusual substrate selectivity code but has not yet been biochemically studied. On the basis of sequence and homology model analysis, we reannotate AmbG as a fatty acyl:adenylate ligase (FAAL)-acyl carrier protein didomain with unusual substrate specificity. The expected adenylate-forming activity on fatty acids was confirmed by in vitro studies. AmbG also adenylates a number of structurally diverse carboxylic acids, including functionalised fatty acids and unsaturated and aromatic carboxylic acids. HPLC-MS analysis and competition experiments show that AmbG preferentially acylates its ACP with long-chain hydrophobic acids and tolerates a π system and a branch near the carboxylic acid. AmbG is the first characterised example of a FAAL-ACP didomain that is centrally located in a PKS and apparently activates a polyketidic intermediate. This is an important step towards deeper biosynthetic studies such as partial reconstitution of the ambruticin pathway to elucidate DVC formation.

Biosynthesis of Chlorinated Lactylates in Sphaerospermopsis sp. LEGE 00249

“…e.g. 41 Identification of Pyruvate as a Precursor of the Lactate Moiety in 1−4. We sought to clarify whether pyruvate would be incorporated directly into the lactate portion of 1−4, as per our biosynthetic proposal.…”

A Priming Cassette Generates Hydroxylated Acyl Starter Units in Mupirocin and Thiomarinol Biosynthesis

“…Here, the ACP of an unusual FAAL-ACP didomain enzyme, AmbG, is proposed to activate a modular (AmbF) ACP-bound polyketide via formation of bis-thioester, effectively cross-linking two PKS synthase components. 32 Homology modeling suggests MupQ/TmlQ share the common ANL family fold characterized by two domains, an N-terminal domain (∼350 aa) and a smaller C-terminal domain (∼100 aa), that undergo significant rearrangements through the catalytic cycle. 23 Conserved sequence motifs have been identified for the ANL superfamily in both the NRPS adenylation domains (A1−10) 29 and for aromatic adenylate forming ligases (Motif I−III).…”

“…As far as we are aware, there is only one similar example of bis-thioester formation recently reported for the ambruticin biosynthetic pathway. Here, the ACP of an unusual FAAL-ACP didomain enzyme, AmbG, is proposed to activate a modular (AmbF) ACP-bound polyketide via formation of bis-thioester, effectively cross-linking two PKS synthase components …”

A universal pocket in Fatty acyl-AMP ligases ensures redirection of fatty acid pool away from Coenzyme A-based activation

“…The ANL superfamily includes enzymes such as the Acyl/Aryl-CoA ligases (ACS or FACLs), Adenylation domains (A-domains) and Luciferases along with the recently identified Fatty acyl-AMP ligases (FAALs). These enzymes are involved in the production of both primary metabolites such as acyl-CoA and secondary metabolites such as antibiotics 1 , complex lipids 2 , cyclic peptides 3 and lipopeptides 4,5 . Basic metabolic pathways such as β-oxidation, membrane biogenesis, post-translational modifications etc., use primary metabolites such as acyl-CoA.…”