Exploring Castellaniella defragrans Linalool (De)hydratase-Isomerase for Enzymatic Hydration of Alkenes.

Matthias Engleder,Monika Müller,Iwona Kaluzna,Martin Schürmann,Daniel Mink,Erich Leitner,Harald Pichler,Anita Emmerstorfer-Augustin

doi:10.3390/molecules24112092

Abstract

Acyclic monoterpenes constitute a large and highly abundant class of secondary plant metabolites and are, therefore, attractive low-cost raw materials for the chemical industry. To date, numerous biocatalysts for their transformation are known, giving access to highly sought-after monoterpenoids. In view of the high selectivity associated with many of these reactions, the demand for enzymes generating commercially important target molecules is unabated. Here, linalool (de)hydratase-isomerase (Ldi, EC 4.2.1.127) from Castellaniella defragrans was examined for the regio- and stereoselective hydration of the acyclic monoterpene β-myrcene to (S)-(+)-linalool. Expression of the native enzyme in Escherichia coli allowed for identification of bottlenecks limiting enzyme activity, which were investigated by mutating selected residues implied in enzyme assembly and function. Combining these analyses with the recently published 3D structures of Ldi highlighted the precisely coordinated reduction–oxidation state of two cysteine pairs in correct oligomeric assembly and the catalytic mechanism, respectively. Subcellular targeting studies upon fusion of Ldi to different signal sequences revealed the significance of periplasmic localization of the mature enzyme in the heterologous expression host. This study provides biochemical and mechanistic insight into the hydration of β-myrcene, a nonfunctionalized terpene, and emphasizes its potential for access to scarcely available but commercially interesting tertiary alcohols.

Highlights

Asymmetric addition of water to monoterpenes is of huge interest, since it enables access to high-value compounds from renewable, inexpensive starting materials [1]
The amount of soluble Ldi obtained with E. coli BL21star (DE3), E. coli Origami, and E. coli LEMO21(DE3) harboring a pMS470 vector was too low for further use independent of the cultivation conditions, as the enzyme was mostly present in inclusion bodies
The highly enantioselective hydration of β-myrcene to (S)-(+)-linalool by Ldi permits production of a tertiary monoterpene alcohol that is only hardly accessible with other enzymes or organic biotransformations [3,4]. Characterization of this intriguing bifunctionalhydratase-isomerase has opened up routes towards its improvement, and revealed challenges for its biocatalytic application

Summary

Introduction

Asymmetric addition of water to monoterpenes is of huge interest, since it enables access to high-value compounds from renewable, inexpensive starting materials [1]. The bifunctional linalool dehydratase-isomerase (Ldi, EC 4.2.1.127) offers access to the desired tertiary monoterpene alcohol (S)-(+)-linalool by reversible (de)hydration of β-myrcene (Scheme 1). (S)-(+)-linalool is isomerized to geraniol in a follow-up reaction catalyzed by the same enzyme [2]. Since the thermodynamic equilibrium of the reactions favor isomerization of geraniol and dehydration of (S)-(+)-linalool, Ldi may confer detoxification of monoterpene alcohols in vivo [2]. This was illustrated by 100- to even 1,000-fold higher reaction rates for geraniol isomerization 25 μ mol min−1 mg−1) and (S)-(+)-linalool dehydration 9 μ mol min−1 mg−1) compared to the reverse reactions

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Conclusion