Apramycin Biosynthesis: Structure and Mechanism of Action of a New-Type Transaldolase AprG from Streptoalloteichus tenebrarius

Abstract

A distinct transaldolase (TAL) termed AprG accounts for the biosynthesis of the octose core of apramycin. AprG transfers a two-carbon unit of a donor substrate such as GalNAc or GlcNAc to the acceptor substrate 6′-oxolividamine, followed by intramolecular cyclization to generate the bicyclic product. In order to understand the molecular mechanism underlying the action of AprG, we determined the crystal structures of AprG in the apo-form and in complex with donor substrates, including GalNAc and GlcNAc. AprG mainly comprises 12 α-helices and forms an (α/α)₆-barrel structure, different from canonical TALs that adopt an (α/β)₈-TIM barrel fold. The complex structures contain Schiff-base intermediates formed between the donor substrates via residue K257, and interaction networks and mutagenesis experiments imply that E316 should be the key residue that directly engages in the catalytic reaction. The Schiff-base intermediates of the two donor substrates exhibit distinct C3-OH interaction networks and angular strain, which might contribute to the preference of AprG toward GalNAc. Notably, various forms of ligands were observed in the AprG_GlcNAc complex, which are proposed to represent different stages during the AprG-catalyzed process. In particular, the ring-form GlcNAc in AprG_GlcNAc, occupying the putative acceptor substrate-binding site, was used to propose the binding pose of the acceptor substrate. Finally, the catalytic reaction mechanism of AprG was also proposed accordingly.