<i>In Silico</i>
            Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

Liu, Long; Deng, Zhuangmei; Yang, Haiquan; Li, Jianghua; Shin, Hyun‐Dong; Chen, Rachel R.; Du, Guocheng; Chen, Jian

doi:10.1128/aem.03045-13

Cited by 47 publications

(43 citation statements)

References 35 publications

(53 reference statements)

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“…For example, our study is in agreement with other studies that thermostability can be significantly enhanced by the cation-π interaction between Lys202 and Trp204 of an alkaline α-amylase from Alkalimonas amylolytica and Lys7 and Trp99 of a tyrosinase from Streptomyces kathirae SC-13031. The double dentate mode of cation-π interactions (Arg89-Trp90 and Arg89-Tyr58) in mutant H58Y, the cation-π interaction Arg113-Tyr71 located on the two adjacent loops in mutant T71Y, and the cation-π interaction Lys330-Tyr304 stabilizing the C-terminal long loop freely swings in mutant T304Y showed a significant increase in apparent T m value of ~3.9 °C, 0.6 °C, and 3.4 °C, respectively.…”

Section: Discussionsupporting

confidence: 93%

Probing the role of cation-π interaction in the thermotolerance and catalytic performance of endo-polygalacturonases

et al. 2016

Sci Rep

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Understanding the dynamics of the key pectinase, polygalacturonase, and improving its thermotolerance and catalytic efficiency are of importance for the cost-competitive bioconversion of pectic materials. By combining structure analysis and molecular dynamics (MD) simulations, eight mutagenesis sites having the potential to form cation-π interactions were identified in the widely used fungal endo-polygalacturonase PG63. In comparison to the wild-type, three single mutants H58Y, T71Y and T304Y showed improved thermostability (the apparent Tms increased by 0.6−3.9 °C) and catalytic efficiency (by up to 32-fold). Chromatogram analysis of the hydrolysis products indicated that a larger amount of shorter sugars were released from the polygalacturonic acid by these three mutants than by the wild-type. MD analysis of the enzyme-substrate complexes illustrated that the mutants with introduced cation-π interaction have modified conformations of catalytic crevice, which provide an enviable environment for the catalytic process. Moreover, the lower plasticity of T3 loop 2 at the edge of the subsite tunnel appears to recruit the reducing ends of oligogalacturonide into the active site tunnel and initiates new hydrolysis reactions. This study demonstrates the importance of cation-π interaction in protein conformation and provides a realistic strategy to enhance the thermotolerance and catalytic performance of endo-polygalacturonases.

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Section: Discussionsupporting

confidence: 93%

Probing the role of cation-π interaction in the thermotolerance and catalytic performance of endo-polygalacturonases

et al. 2016

Sci Rep

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“…As expected, the constructed disulfide bond enhanced the thermostability of azoreductase while the protein secondary structure was unchanged. Similar results were reported for glucose1‐dehydrogenase , alkaline α‐amylase from Alkalimonas amylolytica , lipase B from Candida antarctica , and many other enzymes but to the best of our knowledge; there is no such report on azoreductases.…”

Section: Discussionsupporting

confidence: 85%

“…In recent years, attempts to enhance the kinetic and stability parameters of azoreductases have led to engineering of enzymes with higher thermostability , changing the substrate selectivity , or shifting the optimal pH value . Engineering of disulfide bonds in proteins is a common strategy in rational design, which has been applied to improve the thermostability of various enzymes, such as alkaline α‐amylase . Disulfide bonds can lower the total entropy of the protein, thus slowing the reversible unfolding processes of proteins .…”

Section: Introductionmentioning

confidence: 99%

Improving the thermal stability of azoreductase from Halomonas elongata by introducing a disulfide bond via site‐directed mutagenesis

Nakhaee

Asad

Khajeh

et al. 2018

Biotech and App Biochem

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Azoreductases mainly reduce azo dyes, the largest class of colorants, to colorless aromatic amines. AzoH, a new azoreductase from the halophilic bacterium, Halomonas elongata, has been recently cloned and expressed in Escherichia coli. The aim of this study was to improve thermal stability of this enzyme by introducing new disulfide bonds. Since X-ray crystallography was not available, homology modeling and molecular dynamics was used to construct the enzyme three-dimensional structure. Potential disulfide bonds for increasing thermal stability were found using DIScover online software. Appropriate mutations (L49C/D108C) to form a disulfide bond were introduced by the Quik-Change method. Mutant protein expressed in E. coli showed increased thermal stability at 50 °C (increased half-life from 12.6 Min in AzoH to 26.66 Min in a mutated enzyme). The mutated enzyme could also tolerate 5% (w/v) NaCl and retained 30% of original activity after 24 H incubation, whereas the wild-type enzyme was completely inactivated. According to circular dichroism studies, the secondary structure was not altered by this mutation; however, a blue shift in intrinsic florescent graph revealed changes in the tertiary structure. This is the first study to improve thermal stability and salt tolerance of a halophilic azoreductase.

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“…We further analyzed whether there were changes of other factors besides disulfide bonds in the wild-type and variant enzymes with Discovery Studio (8). The result showed no variation of the amount and distribution of hydrogen bonds (803 pairs) and salt bridges (57 pairs), and the properties of variants under reducing conditions (the disulfide bonds were broken by the reducing reagent DTT) were similar to those of wild-type Cbo FDH (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…Thus, the replacement of the free cysteine residues of FDHs is not the best strategy to improve enzyme chemical stability. As a feasible strategy, the construction of disulfide bonds is widely applied in improving the thermal stability of enzymes such as α-amylase (8), xylanases (9), alkaline protease (10), and lipases (11). There are no reports that disulfide bonds can be formed in wild-type FDHs.…”

Section: Introductionmentioning

confidence: 99%

Elimination of a Free Cysteine by Creation of a Disulfide Bond Increases the Activity and Stability of Candida boidinii Formate Dehydrogenase

Zheng

Yang

Zhou

et al. 2017

Appl Environ Microbiol

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NAD+-dependent formate dehydrogenase (FDH; EC 1.2.1.2) is an industrial enzyme widely used for NADH regeneration. However, enzyme inactivation caused by the oxidation of cysteine residues is a flaw of native FDH. In this study, we relieved the oxidation of the free cysteine of FDH from Candida boidinii (CboFDH) through the construction of disulfide bonds between A10 and C23 as well as I239 and C262. Variants A10C, I239C, and A10C/I239C were obtained by the site-directed mutagenesis and their properties were studied. Results showed that there were no significant changes in the optimum temperature and pH between variants and wild-type CboFDH. However, the stabilities of all variant enzymes were improved. Specifically, the CboFDH variant A10C (A10Cfdh) showed a significant increase in copper ion resistance and acid resistance, a 6.7-fold increase in half-life at 60°C, and a 1.4-fold increase in catalytic efficiency compared with the wild type. Asymmetric synthesis of l-tert-leucine indicated that the process time was reduced by 40% with variant A10Cfdh, which benefited from the increase in catalytic efficiency. Circular dichroism analysis and molecular dynamics simulation indicated that variants that contained disulfide bonds lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity without affecting the secondary structure of enzyme. This work is expected to provide a viable strategy to avoid the microbial enzyme inactivation caused by the oxidation of the free cysteine residues and improving their performances.IMPORTANCE FDH is widely used for NADH regeneration in dehydrogenase-based synthesis of optically active compounds to decrease the cost of production. This study highlighted a viable strategy that was used to eliminate the oxidation of free cysteine residues of FDH from Candida boidinii by the introduction of disulfide bonds. Using this strategy, we obtained a variant FDH with improved activity and stability. The improvement of activity and stability of FDH is expected to reduce its price and then further to decrease the cost of its application.

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In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

Cited by 47 publications

References 35 publications

Probing the role of cation-π interaction in the thermotolerance and catalytic performance of endo-polygalacturonases

Probing the role of cation-π interaction in the thermotolerance and catalytic performance of endo-polygalacturonases

Improving the thermal stability of azoreductase from Halomonas elongata by introducing a disulfide bond via site‐directed mutagenesis

Elimination of a Free Cysteine by Creation of a Disulfide Bond Increases the Activity and Stability of Candida boidinii Formate Dehydrogenase

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