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

Abstract

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.

Structural and Functional Insight into the Mechanism of the Fe−S Cluster‐Dependent Dehydratase from Paralcaligenes ureilyticus

“…Maintaining a stable Fe 3 + species in position Fe1 of the cluster is of benefit for the reaction mechanism since Fe1 is proposed to act as anchor point for the substrate and due to its high Lewis acidity Fe 3 + promotes the removal of the hydroxide group bound to C3 of the substrate. [22] The role of the divalent metal ion in the catalytic mechanism of PuDHT…”

“…[23] Additionally, this bicarbonate interacts with Ser476, which has been proposed to play a crucial role in the reaction mechanism by abstracting the proton from the C2 atom of the substrate. [22] The two bicarbonate ions occupy a position in the active site similar to that of the bound substrate (see below). The presence of the two bicarbonate ions shows there is an access channel that allows them to reach the Fe-S cluster and the vicinity of Lys127.…”

“…The combined interactions involving the functional groups at positions C1 and C2 orient the substrate to facilitate the abstraction of the proton from C2 by Ser476 (distance of 2.9 Å), the initial step of the catalytic cycle that leads to the formation of a carbanion intermediate. [22,25,26] Several additional interactions stabilise the bound D-gluconate in the active site. The hydroxyl groups at C3 and C4 coordinate to Fe2 (2.1 Å and 1.9 Å, respectively) to form a five coordinate square pyramid.…”

“…FeÀ S cluster-dependent dehydratases catalyse reactions in both modules and thus play a central role in the cascade. [6,[10][11][12] These dehydratases include dihydroxy-acid dehydratase (DHAD) from the branched chain amino acid (BCAA) biosynthesis pathway, [19][20][21][22][23][24] an enzyme involved in the de novo synthesis of the BCAAs (i. e., leucine, valine and isoleucine), and sugar acid-specific dehydratases (DHTs), which are involved in the catabolism of D-glucose via the phosphorylative or oxidative Entner-Doudoroff (EDD) pathway. [22][23][24][25][26][27] DHADs and DHTs form the ilvD/EDD superfamily, with ilv referring to the three BCAAs, isoleucine, leucine and valine.…”

“…[6,[10][11][12] These dehydratases include dihydroxy-acid dehydratase (DHAD) from the branched chain amino acid (BCAA) biosynthesis pathway, [19][20][21][22][23][24] an enzyme involved in the de novo synthesis of the BCAAs (i. e., leucine, valine and isoleucine), and sugar acid-specific dehydratases (DHTs), which are involved in the catabolism of D-glucose via the phosphorylative or oxidative Entner-Doudoroff (EDD) pathway. [22][23][24][25][26][27] DHADs and DHTs form the ilvD/EDD superfamily, with ilv referring to the three BCAAs, isoleucine, leucine and valine. However, an alternative classification for these enzymes has recently been proposed that is based on their substrate preferences, leading to three distinct groups instead of the two described above (DHT and DHAD).…”