# Biosynthesis of Nature-Inspired Unnatural Cannabinoidss
## 3 Aromatic Prenyltransferases
### Plant Aromatic Prenyltransferases  
Since the first plant prenyltransferase was characterized and expressed by [PERSON] et al. in 2002, only about 67 plant aromatic prenyltransferases have been identified and functionally characterized [54] in the past decade. Plant aromatic prenyltransferases often show remarkable substrate specificity, catalyzing the regio- and stereospecific prenylation of aromatic substrates. For instance, the prenyltransferase from _Lotus japonicus_ is dimethylallyl diphosphate (DMAPP) and genistein-specific and solely produces 6-prenylgenistein, which has anti-fungal properties [55]. Most of the known plant aromatic prenyltransferases are highly specific to DMAPP as its prenyl donor, although there are a few exceptions. Apart from _Cs_PT1 and _Cs_PT4 from _C. sativa_[20; 53], HilPT1L and HilPT2 involved in bitter acid biosynthesis in _Humulus lupulus_ (hops; a close relative of _Cannabis_), p-hydroxybenzoate geranyltransferases (LePGT-1 and LePGT-2) involved in shikonin biosynthesis in purple gromwell (_Lithospermrum erythorhizon_) [56], were all found to be able to accept GPP as the prenyl donor to transfer the geranyl moiety to a range of aromatic substrates.  
The first plant farnesyltransferase, _Rd_PT1, was discovered in _R. dauricum_, which produces grifolic acid (GFA) from farnesyl pyrophosphate (FPP) and oversellinic acid. Thefarnesyl moiety of GFA can undergo oxidative cyclization to form daurichromenic acid, which has potent anti-HIV and anti-inflammatory activities [57]. OSA is an OLA analogue with a methyl side chain instead of the pentyl side chain at C6 of the resorcinolic acid ring, as previously mentioned. In terms of substrate specificity, _Rd_PT1 was characterized to be highly specific for OSA as its prenyl acceptor, while being highly promiscuous for its prenyl donor, using GPP, FPP and geranylgeranyl pyrophosphate (GGPP), although it generally prefers its native FPP substrate for catalysis. Prenyltransferase such as _Rd_PT1 that are able to use longer chain lengths than GPP, such as the C15 FPP and C20 GGPP, are interesting targets of consideration for the diversification of cannabinoid products in the cannabinoid biosynthetic pathway. The longer prenyl chains of CBGA analogues will allow for more complex cyclization patterns in the following oxidative cyclization step catalyzed by cannabinoid synthases. This will, in turn, generate a new class of novel cannabinoids with C15 or C20 substructures at the C3 position of the OLA resorcinolic ring, which may have potential bioactivities that are vastly distinct from the existing class of phytocannabinoids.  
Unfortunately, plant aromatic prenyltransferases, such as the aforementioned _Rd_PT1, are usually membrane-associated proteins with typically 7-9 transmembrane helices. The difficulty in elucidating high-resolution 3D structures of these membrane-associated enzymes currently limits mechanistic insight into the structure-activity relationship of these prenyltransferases. Not much is known, except that these plant aromatic prenyltransferases possess two conserved aspartate-rich motifs (e.g., NDxxDxxxD) in protein loops 2 and 6 to coordinate divalent cations, which can stabilize and orientate the pyrophosphate group of the donor substrate [54]. On the other hand, some bacterial and fungal prenyltransferases [58; 59; 60] that are soluble, well-characterized and with solved structures, may present a better engineering avenue for the prenyl transfer step in cannabinoid biosynthesis.