Sequential regiospecific gem‐diprenylation of tetrahydroxyxanthone by prenyltransferases from Hypericum sp.

Abstract

Summary Polyprenylated acylphloroglucinol derivatives, such as xanthones, are natural plant products with interesting pharmacological properties. They are difficult to synthesize chemically. Biotechnological production is desirable but it requires an understanding of the biosynthetic pathways. cDNAs encoding membrane‐bound aromatic prenyltransferase (aPT) enzymes from Hypericum sampsonii seedlings (HsPT8px and HsPTpat) and Hypericum calycinum cell cultures (HcPT8px and HcPTpat) were cloned and expressed in Saccharomyces cerevisiae and Nicotiana benthamiana, respectively. Microsomes and chloroplasts were used for functional analysis. The enzymes catalyzed the prenylation of 1,3,6,7‐tetrahydroxyxanthone (1367THX) and/or 1,3,6,7‐tetrahydroxy‐8‐prenylxanthone (8PX) and discriminated nine additionally tested acylphloroglucinol derivatives. The transient expression of the two aPT genes preceded the accumulation of the products in elicitor‐treated H. calycinum cell cultures. C‐terminal yellow fluorescent protein fusions of the two enzymes were localized to the envelope of chloroplasts in N. benthamiana leaves. Based on the kinetic properties of HsPT8px and HsPTpat, the enzymes catalyze sequential rather than parallel addition of two prenyl groups to the carbon atom 8 of 1367THX, yielding gem‐diprenylated patulone under loss of aromaticity of the gem‐dialkylated ring. Coexpression in yeast significantly increased product formation. The patulone biosynthetic pathway involves multiple subcellular compartments. The aPTs studied here and related enzymes may be promising tools for plant/microbe metabolic pathway engineering.

C7-Prenylation of Tryptophan-Containing Cyclic Dipeptides by 7-Dimethylallyl Tryptophan Synthase Significantly Increases the Anticancer and Antimicrobial Activities

“…It has been hypothesized speculated that due to the increase in the hydrophobicity of 2,5-DKPs by adding isoprene groups, the prenylated molecules are better distributed on the membrane than the non-prenylated molecules and are more closely related to the target protein [ 8 , 9 , 35 , 53 ]. In the structures ( 1b – 7b ), the prenyl moieties were connected via its C1 to indole ring C7.…”

“…Mechanistic studies have established that the primary center of DMAPP is attacked by the electron-rich aromatic ring with a concerted displacement of pyrophosphate to form the arenium ion intermediate, which re-aromatizes by deprotonation to form the final product [ 32 , 33 ]. To date, about fifty prenyltransferases from fungi and bacteria belonging to the DMATS super family have been characterized biochemically [ 34 , 35 ]. Structurally, the natural product prenyltransferases share a common structural motif called “ABBA” fold, in which the active site is located in the center of a huge β barrel formed by 10-strand antiparallel β-strands (surrounded by α helices) for protecting the allyl carbanion from the influence of solvents, thereby to promote catalysis [ 36 ].…”

A promiscuous coenzyme A ligase provides benzoyl‐coenzyme A for xanthone biosynthesis in Hypericum

“…Alternatively, as previously suggested for P. hybrida, benzoyl-CoA may be formed in peroxisomes without benzaldehyde formation and then transported to the cytosol through transporters (Qualley et al, 2012). H. calycinum cell cultures accumulate prenylated xanthones upon elicitor treatment (Gaid et al, 2012;Fiesel et al, 2015;Nagia et al, 2019). Benzophenone synthase (BPS) is a cytosolic protein that catalyzes the integration of the benzoyl moiety into the benzophenone core, which is metabolized to xanthones by xanthone synthase, a cytochrome P450 enzyme (Beerhues and Liu, 2009; El-Awaad © 2020 The Authors.…”

“…No amplification products were detected for onekp: BNDE_scaffold_2026015 and onekp:BNDE_scaffold_2009069 with neither Taq-nor high-fidelity DNA polymerases. The HcAAE1 and HcAAE2 ORFs were proofread by Phusion Hot Start II High-Fidelity DNA Polymerase (Fisher Scientific, Schwerte, Germany; https:// www.thermofisher.com) as stated previously (Nagia et al, 2019), except that the T a were 60 and 58°C for HcAAE1 and HcAAE2, respectively. HcAAE1 and HcAAE2 ORFs were digested using NheI/NcoI and NheI/KpnI, respectively.…”

“…Subsequently, C-prenylations are performed by membrane-bound prenyltransferase enzymes. Accumulation of these metabolites in H. calycinum cell cultures is preceded by coordinated increases in the transcript levels of H. calycinum PAL, CNL, BPS, xanthone synthase and prenyltransferase(Gaid et al, 2012;Fiesel et al, 2015;El-Awaad et al, 2016;Nagia et al, 2019)…”