Structure‐Guided Modulation of the Catalytic Properties of [2Fe−2S]‐Dependent Dehydratases

Abstract

The FeS cluster‐dependent dihydroxyacid dehydratases (DHADs) and sugar acid‐specific dehydratases (DHTs) from the ilvD/EDD superfamily are key enzymes in the bioproduction of a wide variety of chemicals. We analyzed [2Fe−2S]‐dependent dehydratases in silico and in vitro, deduced functionally relevant sequence, structure, and activity relationships within the ilvD/EDD superfamily, and we propose a new classification based on their evolutionary relationships and substrate profiles. In silico simulations and analyses identified several key positions for specificity, which were experimentally investigated with site‐directed and saturation mutagenesis. We thus increased the promiscuity of DHAD from Fontimonas thermophila (FtDHAD), showing >10‐fold improved activity toward D‐gluconate, and shifted the substrate preference of DHT from Paralcaligenes ureilyticus (PuDHT) toward shorter sugar acids (recording a six‐fold improved activity toward the non‐natural substrate D‐glycerate). The successful elucidation of the role of important active site residues of the ilvD/EDD superfamily will further guide developments of this important biocatalyst for industrial applications.

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

“…Thus, the sensitivity to inactivation by oxygen and elevated temperatures render both SaDHAD and CjDHAD as unsuitable for biotechnological processes that aim to exploit the catalytic potential of enzymes of the BCAA pathway to manufacture diverse platform chemicals (e.g. isobutanol [12,13,31,32] ). However, both SaDHAD and CjDHAD (and other bacterial DHADs) are suitable targets for the development of much needed new chemotherapeutics to treat infectious diseases.…”

“…DHAD and DHT have been shown to play a central role in cell-free enzyme cascades that facilitate the conversion of glucose to high-value products such as isobutanol. [12,13,[30][31][32] Recent studies have also highlighted DHAD to be an essential enzyme for the growth and survival of microbes and plants; its inhibition by the natural product aspterric acid (K i = 9 nM) greatly impairs the growth of harmful cyanobacteria. [26] Aspterric acid is also a potent inhibitor of both AtDHAD (K i = 0.3 μM) and MtDHAD (K i = 10.1 μM) DHAD.…”

“…[1][2][3][4] The first three enzymes of this pathway are acetohydroxyacid synthase (AHAS) [5] ketol-acid reductoisomerase (KARI) [6][7][8][9] and dihydroxyacid dehydratase (DHAD). [10][11][12][13] AHAS has been used effectively as a target of more than 50 commercial herbicides with some of the inhibitors also showing antimicrobial activity. [3,4] Importantly, sulfometuron methyl, a commercial AHAS herbicide, has been shown to have antituberculosis (TB) activity.…”

“…It belongs to the ilvD/EDD superfamily that also includes sugar acid dehydratases (DHTs). [12,13,23,24] One of the main characteristics of this family is the presence of an FeÀ S cluster in the active site, that is essential for the catalytic function of these enzymes. [23] Two types of FeÀ S clusters have been observed, i.e.…”

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

“…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.…”