The objective of this research was to construct a pydA-pydB hybrid gene that encodes a functional dioxygenase-hydrolase (PydA-PydB) fusion protein for degradation of 3-hydroxy-4-pyridone (HP). HP is an intermediate in both synthesis and degradation of mimosine, a toxic amino acid produced by the tree legume Leucaena leucocephala. Computer-generated models of the fusion proteins suggested that joining of PydA and PydB with 0, 3, or 7 glycine residues as a linker should produce a functional PydA-PydB fusion protein. Accordingly, three hybrid genes, G0, G3, and G7, were constructed in which pydA and pj;dB were connected with 0, 9, and 21 nucleotides, respectively, encoding the glycine residues of the linker region. When these hybrid genes were expressed in Rhizobium and Escherichia coli, only one of them, G3, produced a functional PydA-PydB fusion protein, having both the dioxygenase and hydrolase activities. The G3 hybrid gene could complement both pydA and pydB mutants of Rhizobium, and E coli lysate containing the overexpressed G3 protein was able to degrade HP. This hybrid gene may be useful for developing mimosine-free L. leucocephala plants in the future.