Dihydroxy-Acid Dehydratases From Pathogenic Bacteria: Emerging Drug Targets to Combat Antibiotic Resistance

There is an urgent global need for the development of novel therapeutics to combat the rise of various antibiotic-resistant superbugs. Enzymes of the branched-chain amino acid (BCAA) biosynthesis pathway are an attractive target for novel anti-microbial drug development. Dihydroxy-acid dehydratase (DHAD) is the third enzyme in the BCAA biosynthesis pathway. It relies on an Fe?S cluster for catalytic activity and has recently also gained attention as a catalyst in cell-free enzyme cascades. Two types of Fe?S clusters have been identified in DHADs, i.e. 2Fe?2S] and 4Fe?4S], with the latter being more prone to degradation in the presence of oxygen. Here, we characterise two DHADs from bacterial human pathogens, Staphylococcus aureus and Campylobacter jejuni (SaDHAD and CjDHAD). Purified SaDHAD and CjDHAD are virtually inactive, but activity could be reversibly reconstituted in vitro (up to ～19,000-fold increase with k_cat as high as ～6.7 s^?1). Inductively-coupled plasma-optical emission spectroscopy (ICP-OES) measurements are consistent with the presence of 4Fe?4S] clusters in both enzymes. N-isopropyloxalyl hydroxamate (IpOHA) and aspterric acid are both potent inhibitors for both SaDHAD (K_i=7.8 and 51.6 μM, respectively) and CjDHAD (K_i=32.9 and 35.1 μM, respectively). These compounds thus present suitable starting points for the development of novel anti-microbial chemotherapeutics.