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

Abstract

The traditional way of rationally engineering enzymes to change their biocatalytic properties utilizes the modifications of their active sites. Another emerging approach is the engineering of structural features involved in the exchange of ligands between buried active sites and the surrounding solvent. However, surprisingly little is known about the effects of mutations that alter the access tunnels on the enzymes' catalytic properties, and how these tunnels should be redesigned to allow fast passage of cognate substrates and products. Thus, we have systematically studied the effects of single-point mutations in a tunnel-lining residue of a haloalkane dehalogenase on the binding kinetics and catalytic conversion of both linear and branched haloalkanes. The hotspot residue Y176 was identified using computer simulations and randomized through saturation mutagenesis, and the resulting variants were screened for shifts in binding rates. Strikingly, opposite effects of the substituted residues on the catalytic efficiency toward linear and branched substrates were observed, which was found to be due to substrate-specific requirements in the critical steps of the respective catalytic cycles. We conclude that not only the catalytic sites, but also the access pathways must be tailored specifically for each individual ligand, which is a new paradigm in protein engineering and de novo protein design. A rational approach is proposed here to address more effectively the task of designing ligand-specific tunnels using computational tools.

Filling the Void: Introducing Aromatic Interactions into Solvent Tunnels To Enhance Lipase Stability in Methanol

“…may alter substrate specificity or improve organic solvent resistance (2,(25)(26)(27)(28). The identification of these networks requires computational engines, such as CAVER or MOLE generator, for detecting cavities and tunnels as well as a known structure or model of the target protein (29)(30)(31). The profound work of Damborsky and coworkers on stabilizing haloalkane dehalogenase (DhaA) has demonstrated the potential for the saturation mutagenesis of residues found in solvent channels.…”

“…To date, the solvent tunnel engineering of lipases has not been applied for improving stability in methanol. Most works have aimed to alter enzyme selectivity and substrate specificity (26,31,(51)(52)(53)(54). While the previous report on stabilizing DhaA employed a saturation mutagenesis of tunnel residues (33), herein, a more systematic rational approach was practiced, incorporating bulky aromatic amino acids for introducing new interactions within the LipT6 inner hydrophobic core.…”

Dynamics, a Powerful Component of Current and Future in Silico Approaches for Protein Design and Engineering

“…However, there is still a gap to close, before we can rationally design mutations enhancing ligand transport. In particular, effective means to predict how the ligand presence alters the dynamics of transport pathways to factor in ligand-specific effects of mutations [136] still have to be developed together with more efficient methods to sample the passage of ligands through structural ensembles of proteins.…”

Coarse-grain simulations on NMR conformational ensembles highlight functional residues in proteins

“…Gating residues, lining internal channels connecting a buried active site to the surface, or two distant active sites within a single protein, are a common feature in the protein world, with more than 60% of the enzymes in the Catalytic Site Atlas 12 presenting channels at least 15 Å long 13 , and as shown by the 70 examples discussed in a review by Gora et al 11 . The dynamic properties of these tunnel lining residues play a key role in controlling catalytic efficiency and enzymes substrate specificity [14][15] , and they can be seen as a new category of functional residues, that take a ! 3 part in regulating biological processes without being directly involved in the catalytic reaction.…”