A Stable Artificial Multienzymatic Complex Using a Heterotrimeric Protein From Metallosphaera sedula

Abstract

Bacterial cytochrome P450 monooxygenases (P450s) are promising biocatalysts for chemical syntheses because they catalyze a variety of oxidations on non‐activated hydrocarbons using O₂. However, the requirement of two auxiliary proteins, an electron transfer protein and a reductase, for the catalysis is a major bottleneck for in vitro applications of these monooxygenases. The authors previous study showed that artificial assembly of a bacterial P450 with its auxiliary proteins using a heterotrimeric proliferating cell nuclear antigen (PCNA) from Sulfolobus solfataricus yields a self‐sufficient P450, but partial dissociation of P450 from the complex at catalytic concentrations reduces the apparent specific activity of this self‐sufficient P450. In this study, a Metallosphaera sedula PCNA is used, which is currently the most stable heterotrimeric PCNA, to assemble a bacterial P450 with its auxiliary proteins at submicromolar protein concentrations. The apparent specific monooxygenase activity of the M. sedula PCNA‐assembled P450 with auxiliary proteins is saturated at protein concentrations of 40 nM, and is 2.1‐fold higher than that of the S. solfataricus PCNA‐assembled P450. Therefore, M. sedula PCNA represents a versatile tool to facilitate multiple enzymatic reactions, including the P450 monooxygenase system.