Hydrogen-Bond Network Constraint and Oriented Electric-Field Assistance Cooperatively Govern Selective Oxidative Ring Contraction in Gibberellin Biosynthesis

Gibberellins are best known as plant hormones and are also biosynthesized in bacterial and fungal systems. Their biosynthesis hinges on a remarkable yet mechanistically controversial cytochrome P450−catalyzed oxidative B-ring contraction that converts the 6- 6 - ent -kaurane scaffold into the characteristic 6- 5 - ent -gibberellane scaffold. While pinacol/semi-pinacol rearrangement via a discrete carbocation has long been hypothesized, the precise orchestration of this complex transformation by enzymes such as bacterial CYP114 has remained elusive. Here, we integrate MD simulations with QM/MM calculations to resolve this debate, revealing that CYP114 catalyzes this ring contraction via a concerted, asynchronous proton-coupled electron transfer (PCET)-triggered rearrangement pathway rather than a stepwise pathway involving a discrete carbocation intermediate. More importantly, this transformation is governed by a cooperative control strategy: (i) a clamp-like local hydrogen-bond network that preorganizes the substrate to enforce selective C6−Hβ abstraction while suppressing canonical oxygen rebound, albeit at an energetic cost; and (ii) a strongly oriented internal electric field that compensates for this penalty and selectively stabilizes the charge-redistribution transition state essential for skeletal rearrangement. Comparative analyses of representative fungal and plant homologs suggest that this combination of geometric preorganization and electrostatic assistance may represent a conserved cross-kingdom strategy by which GA-producing P450s promote noncanonical ring contraction. Beyond clarifying the catalytic logic underlying this conserved skeletal remodeling step in gibberellin biosynthesis, these findings reveal how P450s can redirect canonical C−H oxygenation chemistry toward noncanonical oxidative rearrangement.