Crystal Structure\nand Mutagenesis of an XYP Subfamily\nCyclodipeptide Synthase Reveal Key Determinants of Enzyme Activity\nand Substrate Specificity

Cyclodipeptide synthases (CDPSs) catalyze the synthesis\nof diverse\ncyclodipeptides (CDPs) by utilizing two aminoacyl-tRNA (aa-tRNA) substrates\nin a sequential ping-pong reaction mechanism. Numerous CDPSs have\nbeen characterized to provide precursors for diketopiperazines (DKPs)\nwith diverse structural characteristics and biological activities.\nBcmA, belonging to the XYP subfamily, is a cyclo(l-Ile-l-Leu)-synthesizing CDPS involved in the biosynthesis of the\nantibiotic bicyclomycin. The structural basis and determinants influencing\nBcmA enzyme activity and substrate selectivity are not well understood.\nHere, we report the crystal structure of <i>Ss</i>BcmA from <i>Streptomyces sapporonensis</i>. Through structural comparison\nand systematic site-directed mutagenesis, we highlight the significance\nof key residues located in the aminoacyl-binding pocket for enzyme\nactivity and substrate specificity. In particular, the nonconserved\nresidues D161 and K165 in pocket P2 are essential for the activity\nof <i>Ss</i>BcmA without significant alteration of the substrate\nspecificity, while the conserved residues F158 as well as F210 and\nS211 in P2 are responsible for determining substrate selectivity.\nThese findings facilitate the understanding of how CDPSs selectively\naccept hydrophobic substrates and provide additional clues for the\nengineering of these enzymes for synthetic biology applications.