A Structural View on the Stereospecificity of Plant Borneol‐Type Dehydrogenases

Abstract

The development of sustainable processes for the valorization of byproducts and other waste streams remains an ongoing challenge in the field of catalysis. Racemic borneol, isoborneol and camphor are currently produced from α-pinene, a side product from the production of cellulose. The pure enantiomers of these monoterpenoids have numerous applications in cosmetics and act as reagents for asymmetric synthesis, making an enzymatic route for their separation into optically pure enantiomers a desirable goal. Known short-chain borneol-type dehydrogenases (BDHs) from plants and bacteria lack the required specificity, stability or activity for industrial utilization. Prompted by reports on the presence of pure (À )-borneol and (À )-camphor in essential oils from rosemary, we set out to investigate dehydrogenases from the genus Salvia and discov-ered a dehydrogenase with high specificity (E > 120) and high specific activity (> 0.02 U mg À 1 ) for borneol and isoborneol. Compared to other specific dehydrogenases, the one reported here shows remarkably higher stability, which was exploited to obtain the first three-dimensional structure of an enantiospecific borneol-type short-chain dehydrogenase. This, together with docking studies, led to the identification of a hydrophobic pocket in the enzyme that plays a crucial role in the stereo discrimination of bornane-type monoterpenoids. The kinetic resolution of borneol and isoborneol can be easily integrated into the existing synthetic route from α-pinene to camphor thereby allowing the facile synthesis of optically pure monoterpenols from an abundant renewable source.

Rapid high-resolution structure analysis of small, biotechnologically relevant enzymes by cryo-electron microscopy

“…To explore the general applicability of cryo-EM for the high-resolution structure analysis of small plant enzymes, we also subjected SrBDH1 to cryo-EM-based structure analysis. The SrBDH1 preparation yielded a single peak in a SEC/MALS analysis, consistent with a tetramer in solution, in agreement with its crystal structure 11 . As SrBDH1 readily crystallized under various conditions, unlike SoBDH2, we compared the thermal stability of both proteins by differential scanning fluorometry.…”

“…The copyright holder for this preprint this version posted June 15, 2021. ; https://doi.org/10.1101/2021.06.15.448552 doi: bioRxiv preprint used the crystal structure of apo SrBDH1 (PDB ID 6ZZ0) 11 as starting model. The crystal structure and cryo-EM structure are practically identical (Supplementary Table 3).…”

“…BDHs belong to the family of short-chain dehydrogenases-reductases (SDR) 11 . Members of this enzyme class have a TGxxx [AG]xG NAD + -binding motif and a YxxxK active site motif 12,13 and form dimers or tetramers.…”

“…No reuse allowed without permission. 16 ) and the enantioselective BDH from S. rosmarinus (SrBDH1) 11 .…”

Engineering of a borneol dehydrogenase from P. putida for the enzymatic resolution of camphor

“…A tropinone reductaselike short-chain reductase was reported, able to convert pure (+)-camphor and (−)-borneol with a k cat of 0.06 and 0.09, respectively (Reinhardt et al 2014). Recently, an alcohol dehydrogenase from Salvia rosmarinus showed also low activity in the reduction of racemic camphor (Chánique et al 2021). Due to the low activity, these enzymes are unsuitable for the desired kinetic resolution of camphor.…”