Vib-PT, an Aromatic Prenyltransferase Involved in the Biosynthesis of Vibralactone from Stereum vibrans

Abstract

Vibralactone, a hybrid compound derived from phenols and a prenyl group, is a strong pancreatic lipase inhibitor with a rare fused bicyclic β-lactone skeleton. Recently, a researcher reported a vibralactone derivative (compound C1) that caused inhibition of pancreatic lipase with a half-maximal inhibitory concentration of 14 nM determined by structure-based optimization, suggesting a potential candidate as a new antiobesity treatment. In the present study, we sought to identify the main gene encoding prenyltransferase in Stereum vibrans, which is responsible for the prenylation of phenol leading to vibralactone synthesis. Two RNA silencing transformants of the identified gene (vib-PT) were obtained through Agrobacterium tumefaciens-mediated transformation. Compared to wild-type strains, the transformants showed a decrease in vib-PT expression ranging from 11.0 to 56.0% at 5, 10, and 15 days in reverse transcription-quantitative PCR analysis, along with a reduction in primary vibralactone production of 37 to 64% at 15 and 21 days, respectively, as determined using ultra-high-performance liquid chromatography-mass spectrometry analysis. A soluble and enzymatically active fusion Vib-PT protein was obtained by expressing vib-PT in Escherichia coli, and the enzyme’s optimal reaction conditions and catalytic efficiency (Km/kcat) were determined. In vitro experiments established that Vib-PT catalyzed the C-prenylation at C-3 of 4-hydroxy-benzaldehyde and the O-prenylation at the 4-hydroxy of 4-hydroxy-benzenemethanol in the presence of dimethylallyl diphosphate. Moreover, Vib-PT shows promiscuity toward aromatic compounds and prenyl donors. IMPORTANCE Vibralactone is a lead compound with a novel skeleton structure that shows strong inhibitory activity against pancreatic lipase. Vibralactone is not encoded by the genome directly but rather is synthesized from phenol, followed by prenylation and other enzyme reactions. Here, we used an RNA silencing approach to identify and characterize a prenyltransferase in a basidiomycete species that is responsible for the synthesis of vibralactone. The identified gene, vib-PT, was expressed in Escherichia coli to obtain a soluble and enzymatically active fusion Vib-PT protein. In vitro characterization of the enzyme demonstrated the catalytic mechanism of prenylation and broad substrate range for different aromatic acceptors and prenyl donors. These characteristics highlight the possibility of Vib-PT to generate prenylated derivatives of aromatics and other compounds as improved bioactive agents or potential prodrugs.

A versatile cis-prenyltransferase from Methanosarcina mazei catalyzes both C- and O-prenylations

“…Thus, it was surprising that MM_0014, which originally catalyzed C -prenylation on the alkenyl carbon of IPP, could accept simple molecules such as glycerol, ethanol, 1-propanol, 2-propanol, and also H 2 O as acceptor substrates for O -prenylation. Catalysis of both C - and O -prenylation of the same or similar substrates has been reported with some aromatic prenyltransferases ( 21 , 22 , 23 , 24 , 25 ), but it is uncommon in other prenyltransferases such as cPT. The O -prenylation of alcohol is, however, an unlikely physiological reaction for MM_0014.…”

In Depth Natural Product Discovery from the Basidiomycetes Stereum Species

“…Boreostereum vibrans ) and found to inhibit pancreatic lipase with an IC 50 of 0.4 μg mL −1 . It is a lead compound with a novel skeleton structure and is a potential candidate for a new antiobesity therapeutic [ 6 , 7 ]. The absolute configuration of vibralactone (153) was assigned through optical rotation calculation.…”

“…The hirsutane sesquiterpenes (Figure 1) obtained from S. hirsutum, includes hirsutenols A-F (1-6) [9,10], hirsutic acids C-E, N (7-10) [11,12], sterhirsutins A-L (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) [12,13], and sesquiterpenoids 23-25 [14]. Hirsutic acid C (7) was assigned by a combination of chemical and X-ray studies [15]. The absolute configurations of sterhirsutins A (11) and B (12) (assigned as 2R, 3S, 7R, 9S, 11S, 16S, 17R and 2R, 3S, 7R, 9S, 11S, 16R, 17S) were confirmed by X-ray experiments and electronic circular dichroism (ECD) calculations [12].…”

“…In 1985, Schuda et al reported that hirsutic acid C (7) and complicatic acid (27) could be synthesized from the methanoindene in a highly stereoselective manner [25]. In 2006, Kerrie et al reported on the exploitation of cis-1,2-dihydrocatechol in the development of total syntheses of hirsutic acid C (7) and complicatic acid (27) [26,27]. In addition to chemical syntheses, scientists are also working on biosynthetic approaches.…”

“…Secondary metabolites from Stereum had diversiform biological activities, such as cytotoxic, antimicrobial, inhibit pancreatic lipase, nematocidal and so on. Some of them showed promising activities, for example, vibralactone, isolated from Stereum vibrans , is a lead compound with a novel skeleton structure and strong inhibitory activity (IC 50 0.4 μg/mL) against pancreatic lipase, which is a potential candidate for a new antiobesity therapeutic [ 6 , 7 ]. Up to now, there are 10 known species and various unidentified species of Stereum have been investigated for their metabolites.…”