Probing the specificity of CYP112 in bacterial gibberellin biosynthesis

Abstract

Biosynthesis of the gibberellin A (GA) plant hormones evolved independently in plant-associated fungi and bacteria. While the relevant enzymes have distinct evolutionary origins, the pathways proceed via highly similar reactions. One particularly complex transformation involves combined demethylation and γ-lactone ring formation, catalyzed in bacteria by the cytochrome P450 CYP112 in three individual steps, which involves large structural changes in the transition from substrate to product, with further divergence in the recently demonstrated use of two separate mechanistic routes. Here, the substrate specificity of the isozyme from Erwinia tracheiphila, EtCYP112, was probed via UV–Vis spectral binding studies and activity assays with alternate substrates from the GA biosynthetic pathway. EtCYP112 tightly binds its native substrate GA12 and reaction intermediates GA15 and GA24, as well as the methylated derivatives of GA12 and GA15. It, however, only poorly binds methylated GA24, its GA9 final product and the C-20 carboxylate side product GA25. These distinct affinities are consistent with the known reactivity of EtCYP112. However, while it binds to the immediately preceding pathway metabolite GA12-aldehyde and even earlier oxygenated ent-kaurene precursors, EtCYP112 only reacts with GA12-aldehyde and not the earlier ent-kaurene-derived metabolites. Even with GA12-aldehyde conversion is limited to the first two steps, and the full combined demethylation and γ-lactone ring-forming transformation is not catalyzed. Thus, CYP112 has evolved specificity at the catalytic rather than substrate-binding level to enable its role in GA biosynthesis.