Rational Design of Catechol-2, 3-dioxygenase for Improving the Enzyme Characteristics

Abstract

Catechol-2, 3-dioxygenase (C23O) from Pseudomonas sp. CGMCC2953 identified in our laboratory, which is one of the key enzymes responsible for phenanthrene biodegradation, was expected to get better characteristics tolerant to environment for its further application. With the aim of improving the enzyme properties by introducing intermolecular disulfide bonds, X-ray structure of a C23O from Pseudomonas putida MT-2, a highly conserved homologous with the C23O from Pseudomonas sp. CGMCC2953, was directly used to find the potential sites for forming disulfide bonds between two monomers of the target C23O. Two sites, Ala229 and His294, were identified and mutated to cysteine, respectively, by using site mutagenesis. The expected disulfide bond between these two CYS residues was confirmed with both molecular modeling and experimental results. The optimum temperature of the mutated enzyme was widened from 40 to 40 approximately 50 degrees C. The mutated C23O became more alkalescency stable compared with the wild-type enzyme, e.g., 75% of the maximal enzyme activity retained even under pH 9.5 while 50% residue for the wild-type one. Improvement of thermostability of the mutated C230 with the redesigned disulfide was also confirmed.

Activity of a Carboxyl-Terminal Truncated Form of Catechol 2,3-Dioxygenase fromPlanococcussp. S5

“…Activity of this mutant protein at pH 5.0 and pH 4.0 relative to its activity at pH 7.0 was 144.7% and 124%, respectively [22]. In contrast, mutated C23O from Pseudomonas strain became more alkalescency stable compared with the wild-type enzyme and retained 75% of the maximal enzyme activity even under pH 9.5 [37]. …”

“…Most of the examined C23Os show maximal activity at 30–40°C [35, 37–39]. The temperature optimum of the reaction rate of catechol 2,3-dioxygenase from Pseudomonas putida GJ31 was estimated to be 50°C.…”

“…For example, replacement of Ala229 and His294 in catechol-2,3-dioxygenase from Pseudomonas sp. CGMCC2953 by cysteine resulting in formation of additional disulfide bond widened the optimum temperature of such mutated enzyme from 40°C to 40–50°C [37]. The proline rule for thermostabilizing proteins has been proposed by Suzuki et al [41].…”

Cloning and Mutagenesis of Catechol 2,3-Dioxygenase Gene from the Gram-Positive <b><i>Planococcus</i></b> sp. Strain S5

“…To date, many C23Os genes have been cloned from various Gram-negative and Gram-positive bacteria species, including Pseudomonas [Ibrahim, 2003;Jiang et al, 2004;Wei et al, 2010], Comamonas [Parales et al, 1997], Acinetobacter [Takeo et al, 2007], Alcaligenes [Zhu et al, 2008], Bacillus [Duffner and Müller, 1998;Milo et al, 1999], Rhodococcus [Candidus et al, 1994]. However, this study is the first to clone C23O from the genus Planococcus .…”

Protein Engineering Applications on Industrially Important Enzymes: Candida methylica FDH as a Case Study

“…This is followed by experimental construction and analysis of the properties of the designed protein. Besides the improvement of several enzyme properties like coenzyme and substrate specifity (Chul Lee et al, 2009), stability towards to oxidative stress , rational protein design has also been applied to improving the thermostability of several cases Spadiut et al, 2009;Voutilainen et al, 2009;Wei et al, 2009).…”