Activation-Independent Cyclization of Monoterpenoids

Abstract

The biosynthesis of cyclic monoterpenes (C 10 ) generally requires the cyclization of an activated linear precursor (geranyldiphosphate) by specific terpene cyclases. Cyclic triterpenes (C 30 ), on the other hand, originate from the linear precursor squalene by the action of squalene-hopene cyclases (SHCs) or oxidosqualene cyclases (OSCs). Here, we report a novel terpene cyclase from Zymomonas mobilis (ZMO1548-Shc) with the unique capability to cyclize citronellal to isopulegol. To our knowledge, ZMO1548-Shc is the first biocatalyst with diphosphate-independent monoterpenoid cyclase activity. A combinatorial approach using site-directed mutagenesis and modeling of the active site with a bound substrate revealed that the cyclization of citronellal proceeds via a different mechanism than that of the cyclization of squalene.T erpenoids are widespread secondary metabolites present in nearly all organisms. They have important or even essential functions in metabolism and act, for example, as membrane stabilizers, hormones, vitamins, or photoactive compounds. Terpenes are composed of isoprene units that are synthesized either via the mevalonate pathway (13) or via the 1-deoxy-D-xylulose-5-phosphate (DXP) route (12). The common linear precursor of all monoterpenes and higher terpenoids is geranyldiphosphate (30), independent from the pathway used for the synthesis of the isoprene backbone (isopentenyldiphosphate). Terpenoids are present in nature in a large variety of both linear and cyclic forms. The formation of the ring structure of cyclic terpenoids from diphosphate-activated linear precursors is catalyzed by terpene cyclases (terpene synthases) that are specific for either geranyldiphosphate (C 10 ) (monoterpene cyclases), farnesyldiphosphate (C 15 ) (sesquiterpene cyclases), or geranyl-geranyldiphosphate (C 20 ) (diterpene cyclases). Activation by diphosphate is essential for all cyclization reactions catalyzed by class I terpene cyclases. For all currently known diphosphate-dependent cyclizations, the reaction requires the Lewis acid (Mg 2ϩ )-catalyzed ionization of the diphosphate ester of the respective precursor. After isomerization and the subsequent elimination of the diphosphate group, a primary carbocation is generated, which subsequently attacks a nearby double bond, leading to the formation of the more stable carbenium ion (R 3 C ϩ -). Finally, the reaction is terminated by deprotonation, leading to a vast variety of cyclic terpenoids (1, 5, 6).Cyclic triterpenes, such as hopanoids in bacteria or sterols in eukaryotes, arise either from the nonactivated linear precursor squalene (C 30 ) or from 2,3-oxidosqualene. The polycyclization reaction is catalyzed by squalene-hopene cyclases (SHCs), lanosterol synthases, or oxidosqualene cyclases, respectively (21, 25). The cyclization reaction of squalene involves the concerted formation of (i) five ring structures, (ii) 13 carbon-carbon bonds, and (iii) nine stereocenters, rendering it one of the most complex single-step reactions known in biochemistry. Remarka...

Squalene-hopene cyclases—evolution, dynamics and catalytic scope

“…Hoshino and 76 Catalysis and regulation coworkers observed epoxide ring opening with a subsequent cyclization reaction to a drimane skeleton [40]. In addition, Siedenburg and coworkers showed that the SHC from Zymomonas mobilis was able to convert citronellal rac-22 in a Prins-type reaction (WT 30% conversion and W555Y >70% conversion) [41,42]. The cyclization of rac-22 citronellal to isopulegol is an important step in the synthesis of menthol.…”

Expanding the Enzyme Universe: Accessing Non‐Natural Reactions by Mechanism‐Guided Directed Evolution

“…57 Siedenburg et al . conducted site-saturation mutagenesis on three residues in the active site of SHC from Z. mobilis (ZMO1548) to improve production of isomers of isopulegol starting from racemic citronellal (Figure 12B).…”

Enzymatic Addition of Alcohols to Terpenes by Squalene Hopene Cyclase Variants

“…In contrast to the cyclization of farnesol, the incubation of geraniol with purified recombinant Aac SHC wild type afforded a very small amount of its cyclization product. This may reflect the fact that the active site of the enzyme does not favour reactive binding of the geraniol substrate . Our previous studies suggested that alteration of the active site pocket of Aac SHC facilitated the conversion of truncated terpene‐like substrates without disturbing the important catalytic machinery …”