Computational tools in rational metalloenzyme design

Biocatalytic processes are by far provide the best eco-friendly and sustainable ways to synthesize fine chemicals. Metalloenzymes are known to catalyse chemical reactions with exceptionally high regio- and enantioselectivity and with faster reaction kinetics. Metalloenzymes become more desirable than conventional chemical catalysts for the chemical transformation of substrates having multiple functional groups. Due to their high selectivity, metalloenzymes perform chemical modification at a particular site without hampering other more reactive functional groups on the substrates. One limitation of metalloenzymes is that they show a narrow substrate range and often do not catalyse the transformation of non-native substrates. Recent advancements in the field of protein engineering, such as 'directed evolution' and 'rational enzyme design', have substantially expanded the substrate horizon of metalloenzymes. This article describes computationally assisted enzyme design to expand the substrate scope of metalloenzymes. We have taken Cytochrome P450 as a model enzyme to explain the tools frequently used in rational design. We have discussed multiple sequence alignment, tunnels and channels analysis and molecular docking with detailed protocols. This article serves as a step-by-step guide for students learning these concepts.