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There is an urgent global need for the development of novel therapeutics to combat the rise of various antibioticresistant 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 FeÀ S cluster for catalytic activity and has recently also gained attention as a catalyst in cell-free enzyme cascades. Two types of FeÀ S clusters have been identified in DHADs, i.e. [2FeÀ 2S] and [4FeÀ 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 À 1 ). Inductively-coupled plasma-optical emission spectroscopy (ICP-OES) measurements are consistent with the presence of [4FeÀ 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.

Enzyme-catalyzed reaction cascades play an increasingly important role for the sustainable manufacture of diverse chemicals from renewable feedstocks. For instance, dehydratases from the ilvD/EDD superfamily have been embedded into a cascade to convert glucose via pyruvate to isobutanol, a platform chemical for the production of aviation fuels and other valuable materials. These dehydratases depend on the presence of both a FeÀ S cluster and a divalent metal ion for their function. However, they also represent the rate-limiting step in the cascade. Here, catalytic parameters and the crystal structure of the dehydratase from Paralcaligenes ureilyticus (PuDHT, both in presence of Mg 2 + and Mn 2 + ) were investigated. Rate measurements demonstrate that the presence of stoichiometric concentrations Mn 2 + promotes higher activity than Mg 2 + , but at high concentrations the former inhibits the activity of PuDHT. Molecular dynamics simulations identify the position of a second binding site for the divalent metal ion. Only binding of Mn 2 + (not Mg 2 + ) to this site affects the ligand environment of the catalytically essential divalent metal binding site, thus providing insight into an inhibitory mechanism of Mn 2 + at higher concentrations. Furthermore, in silico docking identified residues that play a role in determining substrate binding and selectivity. The combined data inform engineering approaches to design an optimal dehydratase for the cascade.

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