Hydrophobic\nResidues Promote Interfacial Activation\nof Candida rugosa Lipase: A Study of\nRotational Dynamics

Microbial lipases constitute a class of biocatalysts\nwith the ability\nto cleave ester linkages of long-chain triglycerides. This property\nmakes them particularly attractive for industrial applications ranging\nfrom food processing to pharmaceutical preparation. Among such enzymes, Candida rugosa lipase (CRL) is one of the most frequently\nused in biotransformation. A notable feature of CRL, among many lipases,\nis its propensity for interfacial activation: these enzymes exhibit\nelevated catalytic rates when acting at the interface between aqueous\nand hydrophobic phases. Notably, this phenomenon can be attributed\nto the presence of a mobile lid domain, which in its closed state\noccludes the enzyme active site. To advance our understanding of interfacial\nactivation, we explore the dynamics of CRL rotation at the octane–water\ninterface in this work. To do so, we employ molecular dynamics and\numbrella sampling to evaluate the free energy of rotation of the enzyme\nat the interface. We identify a global minimum in the rotational landscape\nthat coincides with lid opening at the interface. Additionally, we\ninvestigate the role of surface residues outside the lid domain as\nthey serve to instigate rotation of the lid toward the aqueous phase.\nIn doing so, we identify a patch of leucine residues which when mutated\nto glycine impose a barrier to rotation that maintains the enzyme\nin the inactive (closed lid) state on the order of 1 μs. Importantly,\nthis study presents a novel quantification of the rotational free\nenergy corresponding to CRL lid opening at the octane–water\ninterface. The accompanying mutagenesis study likewise clarifies the\nrole of hydrophobic surface residues in the transition. As such, this\nwork provides valuable insight into the phenomenon of interfacial\nactivation that might open up new avenues for manipulating the microenvironment\nof industrially relevant lipases, affording enhanced control over\nthe enzyme state.