Structural And Functional Insights Into Asymmetric Enzymatic Dehydration Of Alkenols
Bettina M. Nestl3, Christopher Geinitz3, Stephanie Popa1, Sari Rizek2, Robert J. Haselbeck2, Rosary Stephen2, Michael A. Noble2, Max-Philipp Fischer3, Erik C. Ralph2, Hoi Ting Hau1, Henry Man1, Muhiadin Omar1, Johan P. Turkenburg1, Stephen van Dien2, Stephanie J. Culler2, Gideon Grogan1 & Bernhard Hauer3*
Enzymes are enjoying increasing interest in the chemical industry. Water-adding/removing enzymes such as oleate hydratase, fumarase, and enoyl-CoA hydratase with their special properties demonstrate their potential value in biocatalysis. Dehydratase enzymes that catalyze the regio- and stereoselective dehydration reactions of interest are needed when seeking potential for the production of industrially important conjugated dienes, e.g. butadiene or isoprene as well as tertiary alcohols. Previous investigations addressing the role of LinD in the anaerobic degradation of monoterpenes evaluated its ability to selectively hydrate β-myrcene and to interconvert produced linalool into geraniol. We employed an unbiased approach to evaluate the substrate specificity of the linalool dehydratase isomerase LinD. We confirmed the bifunctional role of LinD in the isomerization of geraniol and derivatives thereof, and the selective dehydration of a broad set of tertiary alcohols. Kinetic resolution of linalool and synthetic analogues by LinD-catalyzed dehydration provided chiral products with selectivity factors exceeding 200. Due to the sterically demanding structure of tertiary alcohols, the synthesis of optically pure tertiary alcohols is still challenging and is achieved via hydrolase-catalyzed kinetic resolution of these compounds. In fact, biotransformations with linalool enantiomers revealed that the (R)-enantiomer was not converted by LinD.
The isomerization activity of LinD was slightly disfavoured compared to the dehydration activity. We assumed the catalytically important cysteine residues in the active site and their oxidation state to be essential for the bifunctionality of the enzyme. The results from our experiments showed that LinD converted various linalool analogues and derivatives thereof reflecting its relatively broad substrate specificity. Furthermore, through our biocatalytic investigations we were the first to demonstrate that substrates accepted by LinD required a specific a-methyl allyl alcohol signature motif. However, the addition of methyl groups onto the linalool methyl and allyl substituent of the signature motif resulted in two- and three-fold reduced conversions. Our studies on the substrate specificity of LinD indicated that linalool analogues with variations of the nature of the substrate (carbon chain length and double bond) significantly influenced the dehydration activity. Interestingly, the product of the C5 substrate, isoprene is currently used in the industrial production of synthetic rubber. Further, we showed that LinD accepted also aromatic substituents and ether analogues of linalool. The significant decrease in activity with linalool analogues modified at the specific signature motif suggested important contributions from active site amino acids to hydrogen-bonding and intermediate stabilization. We assumed that substrates either interfered with the active site preventing a productive binding or that reasonably well bound substrates were difficult to protonate due to a decreased electron density. The attempt to exchange the hydroxyl moiety of linalool with an amine moiety resulted in complete inactivation of LinD.
Crystallization of native LinD and SeMet-LinD allowed a more detailed structural analysis of this catalyst. The architecture of the putative active site allowed us to propose a mechanism for the dehydration and isomerization of monoterpenes linalool and geraniol. We obtained insights into a unique active site that harbours two distinct activities, each using the same amino acids to dehydrate or isomerize monoterpene substrates. The elucidated LinD crystal structures together with site-directed mutagenesis of active site residues and functional characterization allowed us to draw conclusions regarding some features of the reaction mechanism of this unique enzyme: (a) Cys171 and Tyr45/Asp39 as a general acid/base for the protonation of the leaving hydroxyl group of linalool and dehydration at the chiral carbon, (b) activation of water by His129 or Cys180 and addition to the covalent or carbocation intermediate species and (c) formation of either a carbocation intermediate or covalent intermediate between Cys180 and the diene structure. The diene was deprotonated to form myrcene or hydrolyzed triggering the formation of geraniol. Future structural studies will allow the determination of the basis for the unique substrate spectrum of LinD. Based on these findings, LinD provides an exciting opportunity for structure-guided enzyme engineering, particularly in the realization of new biosynthetic pathways in order to gain access to novel industrially interesting diene and tertiary alcohol products.
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