Engineering a de Novo Transport Tunnel

Abstract

Transport of ligands between buried active sites and bulk solvent is a key step in the catalytic cycle of many enzymes. The absence of evolutionary optimized transport tunnels is an important barrier limiting the efficiency of biocatalysts prepared by computational design. Creating a structurally defined and functional "hole" into the protein represents an engineering challenge. Here we describe the computational design and directed evolution of a de novo transport tunnel in haloalkane dehalogenase. Mutants with a blocked native tunnel and newly opened auxiliary tunnel in a distinct part of the structure showed dramatically modified properties. The mutants with blocked tunnels acquired specificity never observed with native family members: up to 32 times increased substrate inhibition and 17 times reduced catalytic rates. Opening of the auxiliary tunnel resulted in specificity and substrate inhibition similar to those of the native enzyme and the most proficient haloalkane dehalogenase reported to date (k cat = 57 s −1 with 1,2-dibromoethane at 37 °C and pH 8.6). Crystallographic analysis and molecular dynamics simulations confirmed the successful introduction of a structurally defined and functional transport tunnel. Our study demonstrates that, whereas we can open the transport tunnels with reasonable proficiency, we cannot accurately predict the effects of such change on the catalytic properties. We propose that one way to increase efficiency of an enzyme is the direct its substrates and products into spatially distinct tunnels. The results clearly show the benefits of enzymes with de novo transport tunnels, and we anticipate that this engineering strategy will facilitate the creation of a wide range of useful biocatalysts.

Ancestral Haloalkane Dehalogenases Show Robustness and Unique Substrate Specificity

“…Althought here have been several attempts to modify HLD substrate specificity by engineering their active sites, [63] halide-binding sites, [45] and access tunnels, [75,76] only two studies achieved as hift in HLD substrate specificity. [45,77] Modification of substrate specificity by ASR has been reported for ancestral a-glucosidases, which preferentially convert maltose-like sugars, whereas their present-day coun- ChemBioChem 2017ChemBioChem , 18,1448ChemBioChem -1456 www.chembiochem.org 2017 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim terpartsd egrade isomaltose-like sugars; [15] ancestral aldehyde dehydrogenaseA LDH1/2c onverted small substrates, whereas the present-day enzymes accommodate bulky aldehydes; [78] and ancestral kinases phosphorylated protein substrates containing prolineo ra rginine at the first position. [79] Therefore, ASR clearly represents au seful strategy for the modification of substrate specificity.…”

Impact of the access tunnel engineering on catalysis is strictly ligand‐specific

“…For example, enzymes catalyzing Diels-Alder [24] and Kemp elimination [25] reactions have been successfully designed. Apart from designing active sites in existing protein folds, the introduction of functional de novo tunnels in protein structures is also possible, and can dramatically affect enzymatic catalysis [26]. Thus, both engineering of existing access tunnels and the introduction of de novo tunnels afford numerous possibilities for obtaining more useful and efficient biocatalysts.…”

Directed evolution methods for overcoming trade‐offs between protein activity and stability

“…The importance of rationally designing enzyme libraries with amino acid diversity in specific regions has also been recently demonstrated in a variety of examples that involve optimization of substrate access tunnels of enzyme active sites to improve activity and stability . Several computational tools have been generated for identifying and engineering substrate access tunnels for diverse applications in enzyme engineering, including altering substrate selectivity (see references within a recent review).…”