Mechanistic Insights into Dideoxygenation in Gentamicin Biosynthesis

Sicong Li, Priscila Dos Santos Bury, Fanglu Huang, Junhong Guo, Guo Sun, Anna Reva, Chuan Huang, Xinyun Jian, Yuan Li, Jiahai Zhou, Zixin Deng, Finian J. Leeper*, Peter F. Leadlay*, Marcio V. B. Dias*, and Yuhui Sun*

ACS Catalysis 2021, 11, 19, 12274–12283

https://pubs.acs.org/doi/10.1021/acscatal.1c03508

Abstract

Gentamicin is an important aminoglycoside antibiotic used for treatment of infections caused by Gram-negative bacteria. Although most of the biosynthetic pathways of gentamicin have been elucidated, a remaining intriguing question is how the intermediates JI-20A and JI-20B undergo a dideoxygenation to form gentamicin C complex. Here we show that the dideoxygenation process starts with GenP-catalyzed phosphorylation of JI-20A and JI-20Ba. The phosphorylated products are successively modified by concerted actions of two PLP (pyridoxal 5′-phosphate)-dependent enzymes: elimination of water and then phosphate by GenB3 and double bond migration by GenB4. Each of these reactions liberates an imine which hydrolyses to a ketone or aldehyde and is then reaminated by GenB3 using an amino donor. Importantly, crystal structures of GenB3 and GenB4 have guided site-directed mutagenesis to reveal crucial residues for the enzymes’ functions. We propose catalytic mechanisms for GenB3 and GenB4, which shed light on the already unrivalled catalytic versatility of PLP-dependent enzymes.