Structure Elucidation\nof the Daptomycin Products Generated\nupon Heterologous Expression of the Daptomycin Resistance Gene Cluster <i>drcAB</i>

Recently, a high-level\ndaptomycin (DAP)-resistant Mammaliicoccus sciuri strain (TS92) was identified,\nwhich mediates a 33% decline of DAP when incubated in Mueller-Hinton\n(MH) medium. The genetic background of the DAP resistance in TS92\nis a newly discovered two-gene operon, named <i>drcAB,</i> whose expression was reported to impair the structural integrity\nof DAP, eventually leading to its inactivation. Here, we set out to\nelucidate the chemical nature of <i>drcAB</i>-mediated DAP\nmodification by applying a general unknown comparative screening (GUCS)\napproach in high-resolution mass spectrometry. DAP in MH medium was\nincubated with Staphylococcus aureus strain RN4220_P<sub>xyl/tet</sub>-<i>drcAB,</i> which\ncarries the <i>drcAB</i> operon under control of an inducible\npromoter on a plasmid, and GUCS test and reference samples were obtained\nupon and without <i>drcAB</i> expression. A two-step process\ncatalyzed by DrcAB was discovered, comprising a structural alteration\nof DAP. The mass spectrometric data indicate an N-substitution at\nthe aniline moiety of kynurenine with dehydroalanine and, subsequently,\na cleavage of the ester bond of the DAP core between kynurenine and\nthreonine by means of water. The structures postulated were confirmed\nby comparison of in silico versus measured fragmentation patterns.