The structural diversity of newly discovered polyketides continues to grow. This review summarises the range of structures with single and multiple β-branches and the mechanistic details of each catalytic step, covering literature from 2008 to August 2020.

Mupirocin, a commercially available antibiotic produced by Pseudomonas fluorescens and thiomarinol, isolated from the marine bacterium Pseudoalteromonas sp. SANK 73390, both consist of a polyketide-derived monic acid homologue esterified with either 9-hydroxynonanoic acid (mupirocin, 9HN) or 8-hydroxyoctanoic acid (thiomarinol, 8HO). The mechanisms of formation of these deceptively simple 9HN and 8HO fatty acid moieties in mup and tml respectively remain unresolved. To define starter unit generation the purified mupirocin proteins MupQ, MupS and MacpD and their thiomarinol equivalents (TmlQ, TmlS and TacpD) have been expressed and shown to convert malonylcoenzyme A (CoA) and succinyl-CoA to 3-hydroxypropionoyl (3-HP) or 4-hydroxybutyryl (4-HB) fatty acid starter units respectively via the MupQ/TmlQ catalysed generation of an unusual bis-CoA/acyl carrier protein (ACP) thioester, followed by MupS/TmlS catalysed reduction. Mix and match experiments show MupQ/TmlQ to be highly selective for the correct CoA, MacpD/TacpD were interchangeable but an alternate transacting ACPs from the mupirocin pathway (MacpA/TacpA) or a heterologous ACP (BatA) were non-functional. MupS and TmlS selectivity was more varied and these reductases differed in their substrate and ACP selectivity. The solution structure of MacpD determined by NMR revealed a C-terminal extension with partial helical character that has been shown to be important for maintaining high titres of mupirocin. We generated a truncated MacpD construct, MacpD_T, which lacks this C-terminal extension but retains an ability to generate 3-HP with MupS and MupQ, suggesting further downstream roles in protein-protein interactions for this region of the ACP.

The presence of b-branches in the structure of polyketides that possess potent biological activity underpins the widespread importance of this structural feature.Kalimantacin is ap olyketide antibiotic with selective activity against staphylococci, and its biosynthesis involves the unprecedented incorporation of three different and sequential b-branching modifications.W eu se purified single and multi-domain enzyme components of the kalimantacin biosynthetic machinery to address in vitro how the pattern of b-branching in kalimantacin is controlled. Robust discrimination of enzyme products required the development of ageneralisable assay that takes advantage of 13 CNMR of asingle 13 Clabel incorporated into key biosynthetic mimics combined with favourable dynamic properties of an acyl carrier protein. We report ap reviously unassigned modular enoyl-CoA hydratase (mECH) domain and the assembly of enzyme constructs and cascades that are able to generate eachs pecific b-branch.

The enoyl‐acyl carrier protein reductase enzyme FabI is essential for fatty acid biosynthesis in Staphylococcus aureus and represents a promising target for the development of novel, urgently needed anti‐staphylococcal agents. Here, we elucidate the mode of action of the kalimantacin antibiotics, a novel class of FabI inhibitors with clinically‐relevant activity against multidrug‐resistant S. aureus. By combining X‐ray crystallography with molecular dynamics simulations, in vitro kinetic studies and chemical derivatization experiments, we characterize the interaction between the antibiotics and their target, and we demonstrate that the kalimantacins bind in a unique conformation that differs significantly from the binding mode of other known FabI inhibitors. We also investigate mechanisms of acquired resistance in S. aureus and identify key residues in FabI that stabilize the binding of the antibiotics. Our findings provide intriguing insights into the mode of action of a novel class of FabI inhibitors that will inspire future anti‐staphylococcal drug development.

The enoyl‐acyl carrier protein reductase enzyme FabI is essential for fatty acid biosynthesis in Staphylococcus aureus and represents a promising target for the development of novel, urgently needed anti‐staphylococcal agents. Here, we elucidate the mode of action of the kalimantacin antibiotics, a novel class of FabI inhibitors with clinically‐relevant activity against multidrug‐resistant S. aureus. By combining X‐ray crystallography with molecular dynamics simulations, in vitro kinetic studies and chemical derivatization experiments, we characterize the interaction between the antibiotics and their target, and we demonstrate that the kalimantacins bind in a unique conformation that differs significantly from the binding mode of other known FabI inhibitors. We also investigate mechanisms of acquired resistance in S. aureus and identify key residues in FabI that stabilize the binding of the antibiotics. Our findings provide intriguing insights into the mode of action of a novel class of FabI inhibitors that will inspire future anti‐staphylococcal drug development.