Most of the antibiotics in clinical use to treat human disease are natural products elaborated by microorganisms. A general concept is that antibiotic resistance genes and mechanisms must have coevolved with antibiotic biosynthetic capability for a just-in-time self-protection scheme. Nature has evolved three major ways for avoidance of suicide in antibiotic-producing microbes, which are antibiotic-modifying, target-protecting enzymes and export pumps. Currently, a novel self-defense mechanism in the biosynthesis of Yatakemycin (YTM) was elucidated: a protein with homology to a recently discovered bacterial DNA glycosylase defines a novel member of the alkylated base DNA glycosylase families and represents the first specific base excision repair (BER) system in secondary metabolism, which contributes to the self-defense in YTM-producing microorganism.

　  YTM is the most potent member among antitumor antibiotics belonging to the family of CC-1065 and duocarmycins known to be DNA alkylating agents (IC50 of 3 pM to L1210 cell line).Members of this family have been shown to selectively bind the minor groove of DNA in AT rich sequences and alkylate adenine at the N3 position. YTM exhibits the most potent cytotoxicity against cancer cell lines probably because of its unique “sandwiched” arrangement with a DNA- binding subunit located on each side of the central alkylation subunit, since the alkylation depends on both shape-selective recognition and shape-dependent catalysis. It’s also reported that YTM even selectively alkylates the nucleosomal DNA almost completely occluded by histones. So it is very interesting to figure out how the YTM producer protects itself against this high cytotoxic compound and avoid its autotoxicity to survive. After cloning the biosynthetic gene cluster and proposed the biosynthetic pathway (J. Am. Chem. Soc., 2012, 134, 8831-8840), we next pay more attention to ytkR2, encoding a protein with homology to a recently discovered bacterial DNA glycosylase. Genetic validation in vivo, biochemical assay and mutagenesis studies in vitro revealed that YtkR2 confers resistance to the producer byspecificitily recognizing and cleaving the YTM modified base: the physiological role of YtkR2 was identified as a novel DNA glycosylase that specificitily recognize and remove the N3-YTM-alkyladenine from the DNA through cleaving the N-glycosylic bond to start the BER pathway. Then homology modeling was used to build the structural model of YtkR2-YTM-DNA complex based on the AlkD-DNA complex structure by collaboration with Prof. Ren-Xiao Wang. Further mutagenesis studies suggested that the bottom of the binding site provides a hydrophobic environment for hosting YTM and the hydrophobic residues on the bottom are critical for excision activity of YtkR2. Besides providing self-resistance mechanisms, the DNA repair system detoxicates the cells from damaged stage. This is the first example that the DNA glycosylase was involved in the self-resistance to DNA-targeting antibiotic. Given the fact that studying novel resistant mechanisms existed in microorganisms could benefit the understanding of the evolutionary dynamics of resistance and sensitivity in Nature, the work also provides a glimpse into BER system for microbial secondary metabolism, which suggests that nature may have evolved different mechanisms for self-defense. These results were published inAngew. Chem. Int. Ed.2012,51, 10532, which was highlighted in Nat. Chem. Biol. 2012, 8, 873.