Characterization of MdDOX-Co homologs revealed a novel gibberellin metabolic clade and a single-residue switch dictating the catalytic site.

The metabolism of gibberellin (GA) is generally mediated by GA 2-oxidase (GA2ox) through 2β-hydroxylation. These enzymes belong to the 2-oxoglutarate-dependent dioxygenase (2ODD, DOX) superfamily. However, MdDOX-Co, another 2ODD, was recently found to catalyze atypical GA 12α-hydroxylation. MdDOX-Co belongs to the DOXC41 clade, which is phylogenetically distant from the canonical GA2ox. The evolutionary and structural bases of this unique catalytic site remain unclear. In this study, we investigated the functional divergence of the DOXC41 clade. Through phylogenetic analyses, biochemical experiments with recombinant enzymes, and GC-MS detection, we discovered that MdDOX-Co homologs exhibit striking diversity in their GA hydroxylation sites, branching into two functional types: those possessing 12α-hydroxylation activity and those possessing 2-hydroxylation activity. We performed enzyme structure predictions, molecular docking, and molecular dynamics (MD) simulations to elucidate the mechanisms that dictate this functional diversity. These in silico approaches identified a key residue that distinguishes the two activities. Subsequent biochemical characterization using a synthesized variant demonstrated that a single-residue switch (T214P) shifted the catalytic activity of MdDOX-Co from 12α-hydroxylation to 2β-hydroxylation. Our findings provide new insights into the structural rationale of how a single amino acid dictates the hydroxylation sites of GA in this novel metabolic clade.