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

Catalysts

卷:11

Oksana Degtjarik¹  Iuliia Iermak²  Ivana Kuta Smatanova³  Petra Havlickova³  Tatyana Prudnikova³  Michal Kuty³  Radka Chaloupkova⁴  Jiri Damborsky⁴ 

[1] Department of Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel;

[2] Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;

[3] Faculty of Science, University of South Bohemia in Ceske Budejovice, Branisovska 1760, 37005 Ceske Budejovice, Czech Republic;

[4] Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic;

关键词: bacterial enzyme;    haloalkane dehalogenase;    mutant form;    crystallization;    tertiary structure;    disulfide bond;   

DOI  :  10.3390/catal11010005

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown