Use of Sulfolobus solfataricus PCNA Subunit Proteins to Direct the Assembly of Multimeric Enzyme Complexes

In nature, enzymes often form multienzyme complexes to enhance their catalytic efficiencies and, �moreover, evolve into genetically fused multidomain enzymes. Inspired by a natural fusion cytochrome P450 (P450) containing a monooxygenase domain and a reductase domain, we have developed a heterotrimeric protein-utilized method to form a multienzyme complex composed of a bacterial P450 and its catalytically essential two redox proteins. Three distinct proliferating cell nuclear antigens (PCNAs) from Sulfolobus solfataricu s, each of which can be separately expressed, spontaneously form a heterotrimer. Fusion to the PCNAs enables complex formation of a bacterial P450 and two redox proteins through the self-assembling of the PCNAs and enhances the activity due to efficient electron transfer in the complex. This PCNA-mediated multienzyme complex formation will be available for other multienzyme reactions.