Enzymatic Pyran Formation Involved in Xiamenmycin Biosynthesis

Abstract

The pyran ring is a very common structural unit of many natural, bioactive molecules that are widely found in plants, bacteria, and fungi. However, the enzymatic processes by which many of these pyran-containing molecules are formed are unclear. Herein, we report the construction of the pyran ring catalyzed by the cooperation of a flavin-dependent monooxygenase, XimD, and a SnoaL-like cyclase, XimE, in the biosynthesis of xiamenmycins. XimD catalyzes the formation of an epoxide intermediate that spontaneously transforms to furan and pyran products (43:1) in vitro. XimE then catalyzes the formation of the pyran ring in a 6-endo configuration from the epoxide to yield a benzopyran, xiamenmycin B. Further, we obtained the crystallographic structure of XimE, with and without product, which suggests a synergistic mechanism in which a group of four residues (Y46–Y90–H102–E136) acts cooperatively as the general acid and base. Subsequent structure-based analysis of possible viable substrates indicates that both XimD and XimE exhibit high promiscuity in their catalysis. Overall, this study reveals the mechanism of pyran ring formation in xiamenmycin biosynthesis and demonstrates the potential application of XimD and XimE in the biosynthesis of other benzoheterocycle scaffolds, including furano- and pyranocoumarins.

Synthetic and biosynthetic methods for selective cyclisations of 4,5-epoxy alcohols to tetrahydropyrans

“…Xiamenmycins are benzopyran natural products isolated from Streptomyces xiamenensis 318 with antifibrotic and anti-inflammatory activities. 112 Biosynthesis of the tetrahydropyran ring moiety in xiamenmycins requires the cooperation of a flavin-dependent monooxygenase (XimD) and a SnoaL-like cyclase (XimE). 112 Using in vitro enzymatic assays, He et al demonstrated that the 3-geranyl-4-hydroxybenzoate precursor 203 is converted by XimD to an unstable epoxide intermediate 204, which spontaneously transforms to a benzofuran product 205.…”

“…112 Biosynthesis of the tetrahydropyran ring moiety in xiamenmycins requires the cooperation of a flavin-dependent monooxygenase (XimD) and a SnoaL-like cyclase (XimE). 112 Using in vitro enzymatic assays, He et al demonstrated that the 3-geranyl-4-hydroxybenzoate precursor 203 is converted by XimD to an unstable epoxide intermediate 204, which spontaneously transforms to a benzofuran product 205. Although the extra methyl substituent on the epoxide intermediate may have a directing effect that has been applied in synthetic efforts to favour the THP ring formation (see section 2.1.5 for examples), no THP ring product was reported in this case.…”

“…However, when XimE was added in the assay, the 6-membered ring benzopyran xiamenmycin B 206 was formed as the major product (Scheme 46). 112 Crystallographic structures of XimE, with and without product, suggested a synergistic mechanism in which residues E136, Y46, and H102 are functionally important to catalysis, substrate binding, and structural stabilisation (Scheme 46). Notably, in studies on substrate scope, these two enzymes exhibited high promiscuity capable of producing 14 products with 6 different benzoheterocyclic scaffolds.…”

“…Notably, in studies on substrate scope, these two enzymes exhibited high promiscuity capable of producing 14 products with 6 different benzoheterocyclic scaffolds. 112 In a follow-up study, this pair of substrate-promiscuous enzymes were further employed for combinatorial biosynthesis of a series of benzoheterocyclic derivatives with greater structural diversity. 113 3.2 Formation of THPs from 4,5-epoxy alcohols in which no EH has been identified 3.2.1 Ambruticin.…”

“…Scheme 46 (A) THP ring formation in xiamenmycin biosynthesis; (B) crystal structure of XimE; (C) proposed mechanism of XimE-catalysed pyran ring formation 112. …”