Abstract

Lipases are interfacially activated enzymes that catalyze the hydrolysis of ester bonds and constitute prime candidates for industrial and biotechnological applications ranging from detergent industry, to chiral organic synthesis. As a result, there is an incentive to understand the mechanisms underlying lipase activity at the molecular level, so as to be able to design new lipase variants with tailor-made functionalities. Our understanding of lipase function primarily relies on bulk assay averaging the behavior of a high number of enzymes masking structural dynamics and functional heterogeneities. Recent advances in single molecule techniques based on fluorogenic substrate analogues revealed the existence of lipase functional states, and furthermore so how they are remodeled by regulatory cues. Single particle studies of lipases on the other hand directly observed diffusional heterogeneities and suggested lipases to operate in two different modes. Here to decipher how mutations in the lid region controls Thermomyces lanuginosus lipase (TLL) diffusion and function we employed a Single Particle Tracking (SPT) assay to directly observe the spatiotemporal localization of TLL and rationally designed mutants on native substrate surfaces. Parallel imaging of thousands of individual TLL enzymes and HMM analysis allowed us to observe and quantify the diffusion, abundance and microscopic transition rates between three linearly interconverting diffusional states for each lipase. We proposed a model that correlate diffusion with function that allowed us to predict that lipase regulation, via mutations in lid region or product inhibition, primarily operates via biasing transitions to the active states.

Highlights

  • Lipases are interfacially activated enzymes that catalyze the hydrolysis of ester bonds and constitute prime candidates for industrial and biotechnological applications ranging from detergent industry, to chiral organic synthesis

  • Our recent studies on Thermomyces lanuginosus lipase (TLL) based on this methodology revealed the existence of discrete functional states that were redistributed by allosteric regulation[23]

  • The dynamic exploration of conformational and functional states of lipases has been characterized by us and others at the single turnover level[23,26,31,58], who quantified their dependence on regulatory cues

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Summary

Introduction

Lipases are interfacially activated enzymes that catalyze the hydrolysis of ester bonds and constitute prime candidates for industrial and biotechnological applications ranging from detergent industry, to chiral organic synthesis. We proposed a model that correlate diffusion with function that allowed us to predict that lipase regulation, via mutations in lid region or product inhibition, primarily operates via biasing transitions to the active states Lipases such as the one from Thermomyces lanuginosus (TLL) are degrading fat and the tight regulation of their activity is central for controlling a plethora of vital biological processes. On the other hand, allow extraction of diffusional behaviors from individual molecules, yielding critical insights in protein function[35,36] Such pivotal studies of lipases on native substrate layers provided the first insights on the interaction and diffusional properties of lipases with native substrates and proposed the existence of two diffusional states[37]. The observed redistribution of conformational sampling by mutations and product presence, allowed us to provide correlations of sampling between diffusional states to the overall lipase function regulation

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