Although the N-terminally attached fatty acids are key structural elements of nonribosomally assembled lipopeptide antibiotics, little is known about the mechanism of lipid transfer during the initial step of biosynthesis. In this study, we investigated the activity of the dissected initiation module (C-A(Glu)-PCP) of surfactin synthetase SrfAA in vitro to gain further insights into the lipoinitiation reaction. The dissected condensation (C) domain catalyzes the transfer of CoA-activated 3-hydroxy fatty acid with high substrate specificity at its donor site to the peptidyl carrier protein (PCP) bound amino acid glutamate (Glu(1)). Additionally, biochemical studies on four putative acyl CoA ligases in Bacillus subtilis revealed that two of them activate 3-hydroxy fatty acids for surfactin biosynthesis in vitro and that the disruption of corresponding genes has a significant influence on surfactin production.

The nonheme iron oxygenase VioC from Streptomyces vinaceus catalyzes Fe(II)‐dependent and α‐ketoglutarate‐dependent Cβ‐hydroxylation of l‐arginine during the biosynthesis of the tuberactinomycin antibiotic viomycin. Crystal structures of VioC were determined in complexes with the cofactor Fe(II), the substrate l‐arginine, the product (2S,3S)‐hydroxyarginine and the coproduct succinate at 1.1–1.3 Å resolution. The overall structure reveals a β‐helix core fold with two additional helical subdomains that are common to nonheme iron oxygenases of the clavaminic acid synthase‐like superfamily. In contrast to other clavaminic acid synthase‐like oxygenases, which catalyze the formation of threo diastereomers, VioC produces the erythro diastereomer of Cβ‐hydroxylated l‐arginine. This unexpected stereospecificity is caused by conformational control of the bound substrate, which enforces a gauche(–) conformer for χ1 instead of the trans conformers observed for the asparagine oxygenase AsnO and other members of the clavaminic acid synthase‐like superfamily. Additionally, the substrate specificity of VioC was investigated. The side chain of the l‐arginine substrate projects outwards from the active site by undergoing interactions mainly with the C‐terminal helical subdomain. Accordingly, VioC exerts broadened substrate specificity by accepting the analogs l‐homoarginine and l‐canavanine for Cβ‐hydroxylation.

Biosynthesis of the hydroxamate-type siderophore erythrochelin requires the generation of δ-N-acetyl-δ-N-hydroxy-L-ornithine (L-haOrn), which is incorporated into the tetrapeptide at positions 1 and 4. Bioinformatic analysis revealed the FAD-dependent monooxygenase EtcB and the bifunctional malonyl-CoA decarboxylase/acetyltransferase Mcd to be putatively involved in the generation of L-haOrn. To investigate if EtcB and Mcd constitute a two-enzyme pathway for the biosynthesis of L-haOrn, they were produced in a recombinant manner and subjected to biochemical studies in vitro. Hydroxylation assays employing recombinant EtcB gave rise to δ-N-hydroxy-L-ornithine (L-hOrn) and confirmed the enzyme to be involved in building block assembly. Acetylation assays were carried out by incubating L-hOrn with recombinant Mcd and malonyl-CoA as the acetyl group donor. Substrate turnover was increased by substituting malonyl-CoA with acetyl-CoA, bypassing the decarboxylation reaction which represents the rate-limiting step. Consecutive enzymatic synthesis of L-haOrn was accomplished in coupled assays employing both the L-ornithine hydroxylase and Mcd. In summary, a biosynthetic route for the generation of δ-N-acetyl-δ-N-hydroxy-L-ornithine starting from L-ornithine has been established in vitro by tandem action of the FAD-dependent monooxygenase EtcB and the bifunctional malonyl-CoA decarboxylase/acetyltransferase Mcd.