Structural and Functional Insight into the Mechanism of the Fe−S Cluster‐Dependent Dehydratase from Paralcaligenes ureilyticus. Issue 9 (27th December 2022)

• Record Type: Journal Article
• Authors: Bayaraa, Tenuun; Lonhienne, Thierry; Sutiono, Samuel; Melse, Okke; Brück, Thomas B.; Marcellin, Esteban; Bernhardt, Paul V.; Boden, Mikael; Harmer, Jeffrey R.; Sieber, Volker; Guddat, Luke W.; Schenk, Gerhard
• Is Part Of: Chemistry. Volume 29:Issue 9(2023)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-12-27
• Subjects: biomanufacturing -- branched-chain amino acid biosynthesis -- cell-free enzyme cascades -- dehydratase -- Fe−S cluster -- isobutanol; Chemistry -- Periodicals; 540
• DOI: 10.1002/chem.202203140
• Languages: English
• ISSNs: 0947-6539

Abstract

Abstract: 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 Fe−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 ( Pu DHT, 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 Pu DHT. 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. Abstract : Fe‐S cluster‐dependent dehydratases (DHTs) from the ilvD/EDD superfamily play an central role in enzyme cascades for the biomanufacture of the platform chemical isobutanol. Here, functional data for the DHT from Paralcaligenes ureilyticus have provided unprecedented insight into the catalytic mechanism of these enzymes that may inform bioengineering approaches to optimise the cascade.