Pyridoxal-5′-phosphate-dependent enzyme GenB3 Catalyzes C-3′,4′-dideoxygenation in gentamicin biosynthesis

BACKGROUND: The C-3′,4′-dideoxygenation structure in gentamicin can prevent deactivation by aminoglycoside 3′-phosphotransferase (APH(3′)) in drug-resistant pathogens. However, the enzyme catalyzing the dideoxygenation step in the gentamicin biosynthesis pathway remains unknown. RESULTS: Here, we report that GenP catalyzes 3′ phosphorylation of the gentamicin biosynthesis intermediates JI-20A, JI-20Ba, and JI-20B. We further demonstrate that the pyridoxal-5′-phosphate (PLP)-dependent enzyme GenB3 uses these phosphorylated substrates to form 3′,4′-dideoxy-4′,5′-ene-6′-oxo products. The following C-6′-transamination and the GenB4-catalyzed reduction of 4′,5′-olefin lead to the formation of gentamicin C. To the best of our knowledge, GenB3 is the first PLP-dependent enzyme catalyzing dideoxygenation in aminoglycoside biosynthesis. CONCLUSIONS: This discovery solves a long-standing puzzle in gentamicin biosynthesis and enriches our knowledge of the chemistry of PLP-dependent enzymes. Interestingly, these results demonstrate that to evade APH(3′) deactivation by pathogens, the gentamicin producers evolved a smart strategy, which utilized their own APH(3′) to activate hydroxyls as leaving groups for the 3′,4′-dideoxygenation in gentamicin biosynthesis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-021-01558-7.