Nucleoside analogues are important therapeutic scaffolds, but their synthesis is often protecting-group-intensive. Purine nucleoside phosphorylases offer a mild enzymatic alternative, yet broad substrate accommodation does not necessarily lead to productive glycosyl transfer. Here, we repurposed HpnG, a hopanoid-associated purine nucleoside phosphorylase, through second-shell engineering. Structure-guided substitution of D124 with glycine increased 2-fluoroadenosine formation from 11% to 91% yield and accelerated inosine phosphorolysis by more than 100-fold with little change in the apparent Km for inosine. HpnG-D124G also accepted diverse purine nucleobases and ribose, 2′-deoxyribose, and arabinose donors, enabling access to drug-relevant nucleosides. Molecular dynamics simulations and electronic-structure calculations suggest that D124G enriches donor–acceptor conformations that are properly aligned for glycosyl transfer. These findings establish productive active-site preorganization as a practical strategy for engineering broad-scope nucleoside biocatalysts.
Xu-Xue Liu, De Yang, Rung-Yi Lai et al.· Organic Letters· 0 citations
The symbiotic bacterium Photorhabdus is a rich source of bioactive secondary metabolites that mediate tripartite interactions with nematodes and insect hosts. However, natural products of ribosomal origin remain largely underexplored within this ecological niche. Here, we report the identification of aphotorhaptin A, a darobactin-like peptide (daropeptide) natural product from Photorhabdus asymbiotica, which structurally features an ether crosslink and an N-terminal acetyl unit. Biosynthetic investigation uncovers aphotorhaptin A is matured via an unexpected leader cleavage step, and the subsequent N-terminal acetylation confers metabolic stability that maintains the hexapeptide scaffold integrity. Biochemical and structural studies demonstrate the acetyltransferase PasC exhibits remarkable substrate promiscuity, facilitated by an expansive active-site cavity that accommodates diverse acyl-CoA donors and peptide substrates. Unlike the antimicrobial darobactin, aphotorhaptin A appears to lack antibacterial activity but modulates nematode development, and this activity requires the ether crosslink and the N-terminal acetyl group in the hexapeptide scaffold. These findings expand the chemical and biosynthetic space of ribosomal peptide family and establish its link with nematode development and reproduction.
Suze Ma, Ru Li, Xiang-Yang Gao et al.· Proceedings of the National...· 0 citations
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