Unraveling the Chemical and Enzymatic Logic of Cytokinin Biosynthesis and Inactivation in Fusarium

As a core family of hormone regulating plant growth and development, the biosynthesis and inactivation mechanisms of cytokinins (CKs) have been well studied in plants. However, these processes remain largely uncharacterized in other organisms, particularly fungal phytopathogens. Here, we identify a distinct pathway of CKs from Fusarium and unravel the chemical and enzymatic logic of biosynthesis and inactivation, which mainly includes: (1) the IPT-LOG fusion enzyme FexA catalyzes N6-dimethylallylation of AMP and cleavage of the C1″-N9 bond to form iP (1); (2) the CYP450 FexB not only hydroxylates 1 to generate the canonical plant-type CKs trans-zeatin (tZ, 2) and cis-zeatin (cZ, 3), but also sequentially catalyzes C4'-N6 cyclization to produce a pyrrole derivative (6); (3) the BBE-like oxidase FexC independently mediates oxidative cleavage of compounds 1-3 and unexpectedly converts 1 to C1'-keto-iP (16); (4) FexC and the NmrA-like SDR FexD cooperate to catalyze double bond isomerization, yielding two novel CKs (17 and 19), where FexD functions as a rare NADPH-dependent 1,4-reductase. Biological activity assays demonstrate that the noncanonical fungus-specific compounds 6, 17, and 19 represent new inactive CK forms for plants, whereas 16 exhibits unexpectedly high activity. Importantly, unlike plants, Fusarium employ double bond isomerization and pyrrole formation as novel strategies for CK inactivation. Our findings uncover unusual functions of fungal CK enzymes and reveal the molecular basis underlying CK biosynthesis and inactivation in plant pathogenic fungi, which provide new insights into the discovery and application of CK derivatives.