Late-Stage Tailoring Steps in the Biosynthesis of β-Rubromycin Involve Successive Terminal Oxidations, a Selective Hydroxyl Reduction, and Distinctive O-Methylations

β-Rubromycin (<b>1</b>) has a unique O-methylated naphthoquinone moiety and is an efficient inhibitor of human telomerase. Through <i>in vivo</i> and <i>in vitro</i> investigations, we elucidated the biosynthetic tailoring steps of compound <b>1</b>, which involve the carboxyl terminal via successive oxidizations by RubU and RubO1/RubO2, O-methylation of the carboxyl terminal by RubX, and reduction of C-3' hydroxyl by RubK. The final tautomerization of the naphthoquinone moiety is mediated by RubO, which anchors the transitional intermediate through O-methylation to form the naphthoquinone tautomer. The structure-activity relationship further revealed the significant influences of the terminal modification status on anticancer activities of rubromycins.