Functional Conservation and Divergence of AlpJ-Family Oxygenases Catalyzing C–C Bond Cleavage in Atypical Angucycline Biosynthesis

AlpJ-family
oxygenases catalyze distinctive oxidative B-ring cleavage
and rearrangement reactions during the biosynthesis of atypical angucycline
natural products, which are characterized by unique chemical structures
and diverse biological activities. While the individual functions
of a few AlpJ-family enzymes have been reported, there is a lack of
systematic exploration and functional comparison within this enzyme
family, hindering a comprehensive understanding of the AlpJ-family
oxygenases. In this study, we have systematically explored and analyzed
AlpJ-family oxygenases, identifying 49 representative homologues,
which can be classified into two distinct evolutionary groups. We
revealed that enzymes from different groups exhibit clear functional
differentiation, catalyzing the same angucycline substrate dehydrorabelomycin
into distinct products, whereas enzymes within the same group display
more similar catalytic functions with varying degrees of functional
overlap. This underscores the intriguing functional conservation and
divergence of the AlpJ-family oxygenases. In addition, we report the
first crystal structure of a Group I enzyme, PenE. Structural analysis
and site-directed mutagenesis identified key structural features and
residues within AlpJ-family oxygenases, which harbor hydrophobic substrate-binding
pockets at both the N- and C-termini, both of which are essential
for function. Our findings provide valuable insights into the evolution,
catalytic mechanisms, and functional divergence of this unique family
of oxygenases. Further investigation of these newly identified AlpJ
homologues and their associated biosynthetic gene clusters will facilitate
the discovery of enzymes with unique catalytic mechanisms and bioactive
atypical angucyclines with novel structures.