Structure‐Guided Modulation of the Catalytic Properties of [2Fe−2S]‐Dependent Dehydratases. (23rd March 2022)

• Record Type: Journal Article
• Authors: Melse, Okke; Sutiono, Samuel; Haslbeck, Magdalena; Schenk, Gerhard; Antes, Iris; Sieber, Volker
• Is Part Of: Chembiochem. Volume 23:Number 10(2022)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-03-23
• Subjects: biocatalysts -- bioinformatics -- dehydratases -- enzyme catalysis -- structure-activity relationships; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.202200088
• Languages: English
• ISSNs: 1439-4227

Abstract

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 ( Ft DHAD), showing >10‐fold improved activity toward D‐gluconate, and shifted the substrate preference of DHT from Paralcaligenes ureilyticus ( Pu DHT) 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. Abstract : Enzyme catalysis : In silico and in vitro investigations enabled the elucidation of the role of important active site residues of the enzymes dihydroxyacid dehydratase (DHAD) and sugar acid‐specific dehydratase (DHT), both belonging to the FeS cluster‐dependent ilvD/EDD superfamily. We proposed a new classification for these enzymes, and we were able to alter their substrate preference, improving activities for non‐natural substrates significantly.