Cyclodipeptide synthases, a family of class-I aminoacyl-tRNA synthetase-like enzymes involved in non-ribosomal peptide synthesis

Abstract

Cyclodipeptide synthases (CDPSs) belong to a newly defined family of enzymes that use aminoacyl-tRNAs (aa-tRNAs) as substrates to synthesize the two peptide bonds of various cyclodipeptides, which are the precursors of many natural products with noteworthy biological activities. Here, we describe the crystal structure of AlbC, a CDPS from Streptomyces noursei. The AlbC structure consists of a monomer containing a Rossmann-fold domain. Strikingly, it is highly similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs), especially class-Ic TyrRSs and TrpRSs. AlbC contains a deep pocket, highly conserved among CDPSs. Site-directed mutagenesis studies indicate that this pocket accommodates the aminoacyl moiety of the aa-tRNA substrate in a way similar to that used by TyrRSs to recognize their tyrosine substrates. These studies also suggest that the tRNA moiety of the aa-tRNA interacts with AlbC via at least one patch of basic residues, which is conserved among CDPSs but not present in class-Ic aaRSs. AlbC catalyses its two-substrate reaction via a ping-pong mechanism with a covalent intermediate in which l-Phe is shown to be transferred from Phe-tRNAPhe to an active serine. These findings provide insight into the molecular bases of the interactions between CDPSs and their aa-tRNAs substrates, and the catalytic mechanism used by CDPSs to achieve the non-ribosomal synthesis of cyclodipeptides.

Unravelling the mechanism of non-ribosomal peptide synthesis by cyclodipeptide synthases

“…AlbC synthesizes cyclo(L-Phe-L-Leu) (cFL) and cyclo(L-Phe-L-Phe) (cFF) 2 , and its S37 residue is the target for phenylalanylation 4 ( Fig. 1).…”

“…The aromatic ring (Phe1) of ZPK is buried deep within a hydrophobic pocket, corresponding to that occupied by cyclodithiothreitol in the AlbC structure 4 , or by the buffer components CHES or CAPSO in the structure of YvmC 6 delineated on one side by strands b3, b4 and b6, and on the other side by helix a8. In particular, there is a stacking interaction between F186 and the Phe1 ring (Fig.…”

“…They employ a ping-pong-type catalytic mechanism beginning with the binding of a first aa-tRNA and the transfer of its aminoacyl moiety onto a conserved serine leading to the formation of an aminoacyl enzyme intermediate [4][5][6][7] . The subsequent steps of the catalytic mechanism have not yet been elucidated.…”

“…The crystal structures of three CDPSs have been determined: AlbC from Streptomyces noursei, Rv2275 from Mycobacterium tuberculosis and YvmC from Bacillus licheniformis [4][5][6] . These enzymes possess a common architecture very similar to the catalytic domains of class-I aa-tRNA synthetases (aaRSs), especially class-Ic TyrRSs and TrpRSs.…”

“…These enzymes possess a common architecture very similar to the catalytic domains of class-I aa-tRNA synthetases (aaRSs), especially class-Ic TyrRSs and TrpRSs. In particular, the aminoacyl moiety of the first aa-tRNA substrate of CDPS is thought to bind in a pocket [4][5][6][7] , structurally corresponding to the tyrosine-binding pocket in TyrRSs (PDB 1J1U 8 ). Accordingly, buffer components were identified within this pocket in several CDPS crystal structures 4,6 .…”