title: Enhanced catalytic site thermal stability of cold-adapted esterase EstK by a W208Y mutation.
abstract: Hydrophobic interactions are known to play an important role for cold-adaptation of proteins; however, the role of amino acid residue, Trp, has not been systematically investigated. The extracellular esterase, EstK, which was isolated from the cold-adapted bacterium Pseudomonas mandelii, has 5 Trp residues. In this study, the effects of Trp mutation on thermal stability, catalytic activity, and conformational change of EstK were investigated. Among the 5 Trp residues, W(208) was the most crucial in maintaining structural conformation and thermal stability of the enzyme. Surprisingly, mutation of W(208) to Tyr (W(208)Y) showed an increased catalytic site thermal stability at ambient temperatures with a 13-fold increase in the activity at 40°C compared to wild-type EstK. The structure model of W(208)Y suggested that Y(208) could form a hydrogen bond with D(308), which is located next to catalytic residue H(307), stabilizing the catalytic domain. Interestingly, Tyr was conserved in the corresponding position of hyper-thermophilic esterases EstE1 and AFEST, which are active at high temperatures. Our study provides a novel insight into the engineering of the catalytic site of cold-adapted enzymes with increased thermal stability and catalytic activity at ambient temperatures.
authors.affiliation: Department of Biomedical Science, Daegu University, Gyeongsan 712-714, South Korea; Center for Bio-Nanomaterials, Daegu University, Gyeongsan 712-714, South Korea.
authors.forename: Jerusha
authors.initials: J
authors.lastname: Boyineni
authors.affiliation: Department of Biomedical Science, Daegu University, Gyeongsan 712-714, South Korea; Center for Bio-Nanomaterials, Daegu University, Gyeongsan 712-714, South Korea.
authors.forename: Junyoung
authors.initials: J
authors.lastname: Kim
authors.affiliation: School of Life Sciences and Biotechnology, Kyungpook National University, Daegu 702-701, South Korea.
authors.forename: Beom Sik
authors.initials: BS
authors.lastname: Kang
authors.affiliation: Department of Biomedical Science, Daegu University, Gyeongsan 712-714, South Korea; Center for Bio-Nanomaterials, Daegu University, Gyeongsan 712-714, South Korea.
authors.forename: ChangWoo
authors.initials: C
authors.lastname: Lee
authors.affiliation: Department of Biomedical Science, Daegu University, Gyeongsan 712-714, South Korea; Center for Bio-Nanomaterials, Daegu University, Gyeongsan 712-714, South Korea. Electronic address: shjang@daegu.ac.kr.
authors.forename: Sei-Heon
authors.initials: SH
authors.lastname: Jang
mesh_terms: D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003080:Cold Temperature; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005110:Extracellular Space; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D055550:Protein Stability; D011549:Pseudomonas / Q000737:chemistry* / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D014364:Tryptophan; D004950:Esterases
keywords: Catalytic site; Cold-adapted enzyme; Esterase; Pseudomonas mandelii; Thermal stability