Dataset Information

Stabilization of an ?/?-Hydrolase by Introducing Proline Residues: Salicylic Acid Binding Protein 2 from Tobacco.

ABSTRACT: ?/?-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 ?/?-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 (?T1/2 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 ?T1/2 15min = +25.7 °C. This straightforward proline substitution approach may also stabilize other ?/?-hydrolases.

SUBMITTER: Huang J

PROVIDER: S-EPMC4557962 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Stabilization of an α/β-Hydrolase by Introducing Proline Residues: Salicylic Acid Binding Protein 2 from Tobacco.

Huang Jun J Jones Bryan J BJ Kazlauskas Romas J RJ

Biochemistry 20150709 28

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. Surp ...[more]

PMID: 26110207

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Project description:Earlier studies showed that the artificial elevation of endogenous glutathione (GSH) contents can markedly increase the resistance of plants against different viruses. On the other hand, salicylic acid (SA)-deficient NahG plants display enhanced susceptibility to viral infections. In the present study, the biochemical mechanisms underlying GSH-induced resistance were investigated in various tobacco biotypes displaying markedly different GSH and SA levels. The endogenous GSH levels of Nicotiana tabacum cv. Xanthi NN and N. tabacum cv. Xanthi NN NahG tobacco leaves were increased by infiltration of exogenous GSH or its synthetic precursor R-2-oxo-4-thiazolidine-carboxylic acid (OTC). Alternatively, we also used tobacco lines containing high GSH levels due to transgenes encoding critical enzymes for cysteine and GSH biosynthesis. We crossed Xanthi NN and NahG tobaccos with the GSH overproducer transgenic tobacco lines in order to obtain F1 progenies with increased levels of GSH and decreased levels of SA. We demonstrated that in SA-deficient NahG tobacco the elevation of in planta GSH and GSSG levels either by exogenous GSH or by crossing with glutathione overproducing plants confers enhanced resistance to Tobacco mosaic virus (TMV) manifested as both reduced symptoms (i.e. suppression of hypersensitive-type localized necrosis) and lower virus titers. The beneficial effects of elevated GSH on TMV resistance was markedly stronger in NahG than in Xanthi NN leaves. Infiltration of exogenous GSH and OTC or crossing with GSH overproducer tobacco lines resulted in a substantial rise of bound SA and to a lesser extent of free SA levels in tobacco, especially following TMV infection. Significant increases in expression of pathogenesis related (NtPR-1a, and NtPRB-1b), and glutathione S-transferase (NtGSTtau, and NtGSTphi) genes were evident in TMV-inoculated leaves in later stages of pathogenesis. However, the highest levels of defense gene expression were associated with SA-deficiency, rather than enhanced TMV resistance. In summary, elevated levels of glutathione in TMV-infected tobacco can compensate for SA deficiency to maintain virus resistance. Our results suggest that glutathione-induced redox changes are important components of antiviral signaling in tobacco.

Project description:The enzyme AHAS (acetohydroxy acid synthase), which is involved in the biosynthesis of valine, leucine and isoleucine, is the target of several classes of herbicides. A model of tobacco AHAS was generated based on the X-ray structure of yeast AHAS. Well conserved residues at the herbicide-binding site were identified, and the roles of three of these residues (Phe-205, Val-570 and Phe-577) were determined by site-directed mutagenesis. The Phe-205 mutants F205A, F205H, F205W and F205Y showed markedly decreased levels of catalytic efficiency, and cross-resistance to two or three classes of herbicides, i.e. Londax (a sulphonylurea herbicide), Cadre (an imidazolinone herbicide) and TP (a triazolopyrimidine derivative). None of the mutations caused significant changes in the secondary or tertiary structure of the enzyme. Four mutants of Phe-577, i.e. F577D, F577E, F577K and F577R, showed unaltered V(max) values, but substantially decreased catalytic efficiency. However, these mutants were highly resistant to two or three of the tested herbicides. The three mutants F577D, F577E and F577R had a similar secondary structure to that of wild-type AHAS. Conservative mutations of Phe-577, i.e. F577W and F577Y, did not affect the kinetic properties of the enzyme or its inhibition by herbicides. The mutation Val-570 to Asn abolished the binding affinity of the enzyme for FAD as well as its activity, and also caused a change in the tertiary structure of AHAS. However, the mutant V570Q was active, but resistant to two classes of herbicides, i.e. Londax and TP. The conservative mutant V570I was substantially reduced in catalytic efficiency and moderately resistant to the three herbicides. The results of this study suggest that residues Phe-205, Val-570 and Phe-577 in tobacco AHAS are located at or near the binding site that is common for the three classes of herbicides. In addition, Phe-205 and Val-570 are probably located at the herbicide-binding site that may overlap partially with the active site. Selected mutants of Phe-577 are expected to be utilized to construct herbicide-resistant transgenic plants.

Dataset Information

Stabilization of an ?/?-Hydrolase by Introducing Proline Residues: Salicylic Acid Binding Protein 2 from Tobacco.

Publications

Stabilization of an α/β-Hydrolase by Introducing Proline Residues: Salicylic Acid Binding Protein 2 from Tobacco.

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