X‐ray structure of linalool dehydratase/isomerase from Castellaniella defragrans reveals enzymatic alkene synthesis

Abstract

Edited by Stuart FergusonLinalool dehydratase/isomerase (Ldi), an enzyme of terpene degradation in Castellaniella defragrans, isomerizes the primary monoterpene alcohol geraniol into the tertiary alcohol (S)-linalool and dehydrates (S)-linalool to the alkene b-myrcene. Here we report on the crystal structures of Ldi with and without terpene substrates, revealing a cofactor-free homopentameric enzyme. The substrates were embedded inside a hydrophobic channel between two monomers of the (a,a) 6 barrel fold class and flanked by three clusters of polar residues involved in acid-base catalysis. The detailed view into the active site will guide future biotechnological applications of Ldi, in particular, for industrial butadiene and isoprene production from renewable sources.

On the current role of hydratases in biocatalysis

“…a Active site of Elizabethkingia meningoseptica oleate hydratase (OhyA; PDB-code: 4UIR) with docked oleic acid in grey, FAD in yellow and catalytic residues in light orange (Engleder et al 2015). b Active site of Castelaniella defragrans linalool dehydratase-isomerase (LDI; PDB-code: 5HSS) with co-crystallised β - myrcene in orange and catalytic residues in magenta (Weidenweber et al 2016). c Active site of Pelobacter acetylenicus acetylene hydratase (ACH; PDB-code: 2E7Z) showing both MGD cofactors and the tightly coordinated tungsten (blue), as well as the [4Fe:4S] cluster and the catalytic aspartate (Seiffert et al 2007).…”

“…Since it is commercially hardly available, hydration of β-myrcene was suggested as an intriguing route for ( S )-(+)-linalool production (Lüddeke and Harder 2011; Demming et al 2018), even though its industrial application has not yet been reported. In contrast, LDI is already applied for the production of industrially highly relevant dienes such as isoprene and butadiene (Marliere 2013; Botes and Conradie 2015), which are amounting to market values of billions of dollars annually (Weidenweber et al 2016). …”

“…LDI possesses an N-terminal signal sequence for SEC-dependent periplasmic translocation of the nascent polypeptide. The enzyme is not associated with a cofactor, but sensitive towards molecular oxygen and requires a mild reducing agent for full activity, suggesting that the reduction/oxidation state of its four cysteines are important for full activity (Brodkorb et al 2010; Weidenweber et al 2016). …”

“…Only recently, the crystal structure of C. defragrans LDI was independently solved by the groups of Harder and Hauer, respectively, either in complex with geraniol (Nestl et al 2017) or with geraniol and β-myrcene (Weidenweber et al 2016). In both instances, LDI crystallized as a cyclic homopentamer with a central hole, with each monomer showing an (α,α) 6 barrel fold.…”

“…While this fold is also observed in other proteins with similar functions (Wendt et al 1997), the active site of LDI is located at the interface of two subunits, which is unprecedented among (α,α) 6 barrel proteins. The importance of cysteines for LDI activity was established for an essential disulphide bond capping the substrate channel and for the contribution of the other two cysteines in the putative reaction mechanisms (Weidenweber et al 2016; Demming et al 2017). Both studies independently reported on acid/base catalysis for the (de)hydration and isomerization reactions of LDI, in which ( S )-(+)-linalool is protonated by a cysteine, followed by rehydration for geraniol or deprotonation for β-myrcene (Fig.…”