title: Stabilization of an α/β-Hydrolase by Introducing Proline Residues: Salicylic Acid Binding Protein 2 from Tobacco.
abstract: α/β-Hydrolases are important enzymes for biocatalysis, but their stability often limits their application. We investigated a plant esterase, salicylic acid binding protein 2 (SABP2), as a model α/β-hydrolase. SABP2 shows typical stability to urea (unfolding free energy 6.9 ± 1.5 kcal/mol) and to heat inactivation (T1/2 15min 49.2 ± 0.5 °C). Denaturation in urea occurs in two steps, but heat inactivation occurs in a single step. The first unfolding step in urea eliminates catalytic activity. Surprisingly, we found that the first unfolding likely corresponds to the unfolding of the larger catalytic domain. Replacing selected amino acid residues with proline stabilized SABP2. Proline restricts the flexibility of the unfolded protein, thereby shifting the equilibrium toward the folded conformation. Seven locations for proline substitution were chosen either by amino acid sequence alignment with a more stable homologue or by targeting flexible regions in SABP2. Introducing proline in the catalytic domain stabilized SABP2 to the first unfolding in urea for three of five cases: L46P (+0.2 M urea), S70P (+0.1), and E215P (+0.9). Introducing proline in the cap domain did not stabilize SABP2 (two of two cases), supporting the assignment that the first unfolding corresponds to the catalytic domain. Proline substitutions in both domains stabilized SABP2 to heat inactivation: L46P (ΔT1/2 15min = +6.4 °C), S70P (+5.4), S115P (+1.8), S141P (+4.9), and E215P (+4.2). Combining substitutions did not further increase the stability to urea denaturation, but dramatically increased resistance to heat inactivation: L46P−S70P ΔT1/2 15min = +25.7 °C. This straightforward proline substitution approach may also stabilize other α/β-hydrolases.
authors.affiliation: §School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, People's Republic of China.
authors.forename: Jun
authors.initials: J
authors.lastname: Huang
authors.forename: Bryan J
authors.initials: BJ
authors.lastname: Jones
authors.forename: Romas J
authors.initials: RJ
authors.lastname: Kazlauskas
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry* / Q000235:genetics; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D011392:Proline / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D058767:Protein Unfolding; D014026:Nicotiana / Q000737:chemistry* / Q000235:genetics
chemical_list: D010940:Plant Proteins; D011392:Proline; D006867:Hydrolases; C499506:SABP2 protein, Nicotiana tabacum; D004950:Esterases