Description of Transport Tunnel in Haloalkane Dehalogenase Variant LinB D147C+L177C from Sphingobium japonicum

Abstract

The activity of enzymes with active sites buried inside their protein core highly depends on the efficient transport of substrates and products between the active site and the bulk solvent. The engineering of access tunnels in order to increase or decrease catalytic activity and specificity in a rational way is a challenging task. Here, we describe a combined experimental and computational approach to characterize the structural basis of altered activity in the haloalkane dehalogenase LinB D147C+L177C variant. While the overall protein fold is similar to the wild type enzyme and the other LinB variants, the access tunnels have been altered by introduced cysteines that were expected to form a disulfide bond. Surprisingly, the mutations have allowed several conformations of the amino acid chain in their vicinity, interfering with the structural analysis of the mutant by X-ray crystallography. The duration required for the growing of protein crystals changed from days to 1.5 years by introducing the substitutions. The haloalkane dehalogenase LinB D147C+L177C variant crystal structure was solved to 1.15 Å resolution, characterized and deposited to Protein Data Bank under PDB ID 6s06.

Structural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA

“…These properties make LinB and DmbA a good target for protein modification and bioengineering. A number of these enzyme variants have been successfully crystallized and their crystal structures have been solved [ 19 , 20 , 21 , 22 ].…”

“…The structural and biochemical properties of these tunnels have a major impact on the catalysis and substrate specificity of an enzyme [ 24 ]. Therefore, the access tunnels became a frequent target of enzyme modifications [ 21 ]. Haloalkane dehalogenases contain several tunnels connecting the protein surface and the active site cavity.…”

“…The p2 secondary slot tunnels are served for the alcohol release and water solvent exchange. There are several further variations of tunnel branches originating from p1 and p2 tunnels [ 21 , 23 , 25 ].…”