Mupirocin (Pseudomonic acid A) has long been used against Methicillin Resistant Staphylococcus aureus MRSA, yet bacteria have developed resistance, threatening future use. Structurally similar to mupirocin is thiomarinol A, a natural compound produced by the marine bacterium Pseudoalteromonas spp, which possesses stronger antibacterial activities. However, it differs from mupirocin by four distinct differences and among these are extra 4-hydroxylation and joining to pyrrothine. Studying these differences should enhance our understanding of the molecular assembly and biosynthesis machinery. Complementation and mutagenesis studies identified the tmuB gene to be responsible for the 4-hydroxylation as a final tailoring step. In vivo and in vitro studies on purified TmuB revealed that it can hydroxylate diverse pseudomonic acids but is inhibited by molecules with an 8-hydroxyl group, which primarily affects catalysis rather than binding. Molecular modelling plus docking and mutagenesis provides increased understanding of both TmuB potential to modify other substrates and how mupirocin activity can be modulated by 4-hydroxylation. This study also expressed holA, purified its gene product, a non-ribosomal polypeptide synthetase (NRPS), and assayed its activity by pyrophosphate release. It presents a proposed pathway for pyrrothine biosynthesis catalysed by HolA, which exhibits the unusual ability to join two cysteine molecules by a single NRPS module.