A Single Amino Acid Switch Alters the Prenyl Donor Specificity of a Fungal Aromatic Prenyltransferase toward Biflavonoids

Abstract

Biflavonoids are pharmaceutically important compounds. Prenylation usually improves bioactivity; however, prenylated biflavonoids are rare in nature. Here, we report successful prenylation or geranylation of biflavonoids using fungal prenyltransferase CdpC3PT and its mutants. F253 was identified as a key residue related to donor selectivity, which enables the switching from utilizing DMAPP to GPP precisely at the same C-3′′′ site of biflavonoids. Furthermore, another residue W181 was discovered to generally increase prenylation activity toward biflavonoids.

Dearomative gem-diprenylation of hydroxynaphthalenes by an engineered fungal prenyltransferase

“…CdpC3PT and CdpC3PT_F253G were successfully created, overproduced and purified as described in our previous study. 17 The purity of protein was checked on SDS-PAGE analysis. To test enzyme activities towards hydroxynaphthalenes, the assays (50 μL) containing 50 mM Tris–HCl (pH = 7.5), 1 mM DMAPP, 5 mM Ca 2+ , 5% DMSO, and 50 μg of purified recombinant protein CdpC3PT_F253G or CdpC3PT wild type were incubated with 1 mM of each substrate.…”

“…20 Molecular docking is carried out through the homology model constructed in the previous stage. 17 2D1 was constructed using molecule builder module and energy minimized. The top 15 conformations of 2D1 were docked into the active site of CdpC3PT_F253G or CdpC3PT using an induced fit protocol.…”

“…14,15 Unlike the known enzymatic gem-diprenylation of mono-and tri-cyclic aromatic systems, the enzyme for gem-diprenylation of bi-cyclic hydroxynaphthalenes is new to science. The fungal prenyltransferase CdpC3PT from Neosartorya scheri showed mono-prenylation activities towards several types of substrates such as hydroxynaphthalenes and biavonoids, 16,17 while its mutation on F253 altered donor selectivity towards biavonoids. 17 In this study, we discovered novel gem-diprenylation activities catalyzed by the CdpC3PT_F253G toward a-hydroxynaphthalenes, which expands enzymatic tools for gem-diprenylation of aromatic substrates.…”

“…The fungal prenyltransferase CdpC3PT from Neosartorya scheri showed mono-prenylation activities towards several types of substrates such as hydroxynaphthalenes and biavonoids, 16,17 while its mutation on F253 altered donor selectivity towards biavonoids. 17 In this study, we discovered novel gem-diprenylation activities catalyzed by the CdpC3PT_F253G toward a-hydroxynaphthalenes, which expands enzymatic tools for gem-diprenylation of aromatic substrates. Here we also conrmed the substitution process and explained the alteration of catalytic property aer the mutation.…”

Structure Revision of Balsamisides A–D and Establishment of an Empirical Rule for Distinguishing Four Classes of Biflavonoids

“…However, sometimes it is challenging to assign their planar and stereochemical structures by NMR analysis due to the low H/C ratio in the molecules. This issue is exacerbated when flavonoids are di-, − tri-, − or polymerized or coupled with other scaffolds of natural products, such as simple phenolic compounds, xanthones, , phenylpropanoids, and terpenoids. , Although chemical shift calculations followed by statistical analysis (e.g., CP3, DP4, DP4+) and chiroptical simulation (e.g., ECD, VCD, specific rotation) have been used as powerful methods to propose reliable chemical structures of many natural products, these approaches still require a considerable amount of time, labor, and economic resources. Alternatively, many empirical rules to determine planar structure or relative configuration using diagnostic 1 H and/or 13 C NMR chemical shifts values have been developed and widely used due to their accuracy and convenience. − However, no empirical rule for structural identification of biflavonoids or flavonoid-natural product adducts has yet been reported.…”