WAP-8294A is a family of at least 20 cyclic lipodepsipeptides exhibiting potent anti-MRSA activity. These compounds differ mainly in the hydroxylated fatty acyl chain; WAP-8294A2, the most potent member of the family that reached clinical trials, is based on (R)-3-hydroxy-7-methyloctanoic acid. It is unclear how the acyl group is incorporated because no acyl-CoA ligase (ACL) gene is present in the WAP-8294A gene cluster in Lysobacter enzymogenes OH11. Here, we identified seven putative ACL genes in the OH11 genome and showed that the yield of WAP-8294A2 was impacted by multiple ACL genes with the ACL6 gene having the most significant effect. We then investigated several (R)-3-hydroxy fatty acids and their acyl SNAC (N-acetylcysteamine) thioesters as substrates for the ACLs. Feeding (R)-3-hydroxy-7-methyloctanoate-SNAC to the ACL6 gene deletion mutant restored the production of WAP-8294A2. Finally, we heterologously expressed the seven ACL genes in E. coli and purified six of the proteins. While these enzymes exhibit a varied level of activity in vitro, ACL6 showed the highest catalytic efficiency in converting (R)-3-hydroxy-7-methyloctanoic acid to its CoA thioester when incubated with coenzyme A and ATP. These results provided both in vivo and in vitro evidence to support the fact that ACL6 is the main player for fatty acyl activation and incorporation in WAP-8294A2 biosynthesis. The results also suggest that the molecular basis for the acyl chain diversity in the WAP-8294A family is the presence of functionally overlapping ACLs.
The N-acetylcystamine (SNAC) thioester of dodecapentaenoic acid, an analog of a putative intermediate in the biosynthesis of Heat Stable Antifungal Factor (HSAF), is synthesized. Key steps include sequential Horner-Emmons homologations with the Weinreb amide of diethylphosponoacetic acid, and thioesterification of an aldol-derived 3-hydroxyalkanoate, which serves as a stable precursor of the sensitive polyenoate. The thioester was investigated as a biosynthetic substrate using a purified nonribosomal peptide synthetase and was not incorporated in the observed products.
A simple, sensitive, and rapid chemiluminescence method was developed for the detection and quantification of selected derivatives of artemisinin. It was found that the chemiluminescence (CL) signal resulting from the alkaline luminol reaction of the artemisinin derivatives in the presence of hematin was linear over a wide concentration range. We report results that suggest that a visual test or a test involving digital image capture may be used in addition to a field-ready instrumental approach to combat drug counterfeiting of malarial therapies.
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