Cytochrome P450s are heme-thiolate enzymes that have been broadly applied in pharmaceutical and biosynthesis because of their efficient oxidation at inert carbons. Extensive engineering campaigns are applied to P450s to explore new non-natural substrates and reactions; however, achieving high coupling efficiency is one of the main challenges. The undesirable uncoupling reactions result in the extra consumption of expensive cofactor NAD(P)H, and lead to the accumulation of reactive oxygen species and the inactivation of enzymes and organisms. Using protein engineering methods, these limitations can be overcome by engineering and fine-tuning P450s. A systemic perspective of the enzyme structure and the catalytic mechanism is essential for P450 engineering campaigns for higher coupling efficiency. This review provide an overview on factors contributing to uncoupling and protein engineering approaches to minimize uncoupling and thereby generating efficient and robust P450s for industrials use. Contributing uncoupling factors are classified into three main groups: i) substrate binding pocket; ii) ligand access tunnel(s); and iii) electron transfer pathway(s). Finally, we draw future directions for combinations of effective state-of-the-art technologies and available software/online tools for P450s engineering campaigns.
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