Rational evolution of the unusual Y-type oxyanion hole of Rhodococcus sp. CR53 lipase LipR
Publication date: January 2018 Source:Enzyme and Microbial Technology, Volume 108 Author(s): Belén Infanzón, Pablo H. Sotelo, Josefina Martínez, Pilar Diaz Rhodococcus sp CR-53 lipase LipR was the first characterized member of bacterial lipase family X. Interestingly, LipR displays some similarity with α/β-hydrolases of the C. antartica lipase A (CAL-A)-like superfamily (abH38), bearing a Y-type oxyanion hole, never found before among bacterial lipases. In order to explore this unusual Y-type oxyanion hole, and to improve LipR performance, two modification strategies based on site directed or saturation mutagenesis were addressed. Initially, a small library of mutants was designed to convert LipR Y-type oxyanion hole (YDS) into one closer to those most frequently found in bacteria (GGG(X)). However, activity was completely lost in all mutants obtained, indicating that the Y-type oxyanion hole of LipR is required for activity. A second approach was addressed to modify the two main oxyanion hole residues Tyr110 and Asp111, previously described for CAL-A as the most relevant amino acids involved in stabilization of the enzyme-substrate complex. A saturation mutagenesis library was prepared for each residue (Tyr110 and Asp111), and activity of the resulting variants was assayed on different chain length substrates. No functional LipR variants could be obtained when Tyr110 was replaced by any other amino acids, indicating that this is a crucial residue for catalysis. Ho...
Source: Enzyme and Microbial Technology - Category: Biotechnology Source Type: research