Deoxysugar Formation via 4',5'‐Dehydration on PKS Assembly Line in Nucleoside Antibiotic Biosynthesis

ABSTRACT Amipurimycin is a peptidyl nucleoside antibiotic characterized by a C 9 high‐carbon sugar, with potent activity against the rice blast pathogen Pyricularia oryzae . The biosynthetic machinery of the C‐4' methylene group in its deoxy sugar has remained unknown. This study reveals that ApmL, a hypothetical protein in the DUF3500 family, functions as a 4′,5′‐dehydratase whose activity is strictly dependent on the PKS assembly line. Together with its partner reductase ApmM, they complete a two‐step C‐4′ deoxygenation process via dehydration‐reduction. We achieve the first in vitro reconstitution of this unique PKS‐coupled dehydration process and show direct interaction between ApmL and PKS proteins. Our findings establish ApmL as a new family of dehydratase and uncover a noncanonical deoxygenation strategy in nucleoside antibiotic biosynthesis.