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

β-Rubromycin (1) has a unique O-methylated naphthoquinone moiety and is an efficient inhibitor of human telomerase. Through in vivo and in vitro investigations, we elucidated the biosynthetic tailoring steps of compound 1, 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.