Site-directed mutagenesis of methionine residues for improving the oxidative stability of α-amylase from Thermotoga maritima

Ozturk, Handan; Ece, Selin; Gündeğer, Ersin; Evran, Serap

doi:10.1016/j.jbiosc.2013.04.018

Cited by 18 publications

(6 citation statements)

References 13 publications

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“…Related crystallographic studies have shown that when a substrate (or inhibitor) binds to the active site of αmannosidase, the mannose ring is converted from a stable chair conformation to an unstable boat conformation [32,47], which is required for catalysis and strong interaction with the Zn 2+ cofactor at the active site [24]. The β-mannanases (EC3.2.1.78) mutant screened by Zhang et al by random mutation showed a significant improvement in specific activity and thermal stability compared with the parental one [44]; Ozturk et al found that a-amylase's oxidative stability was significantly increased by changing four methionine-containing sites (43, 44, 55, and 62) on that enzyme to alanine [25]. These reseach further indicate that a slight conformational change appears to influence the substrate-binding and enzyme catalytic characteristics [14].…”

Section: Discussion：mentioning

confidence: 99%

Biochemical characteristics of point mutated <i>Capra hircus</i> lysosome α-mannosidase

Wang

Zhang

Teng

et al. 2023

The Journal of Veterinary Medical Science

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Locoweeds, a type of poisonous weedare, are widely distributed throughout the world and have a significant impact on the development of herbivore animal husbandry. Swainsonine (SW), the main toxin in locoweeds, can competitively inhibit lysosomes α-mannosidase (LAM) in animal cells, resulting in α-mannosidosis. However, the specifics of the interaction between SW and LAM are still unclear. Here, we used molecular docking to predicte the interaction points between SW and LAM, built mutated lysosomes α-mannosidase (LAM M ), and analyzed its biochemical properties changes in presumption points. The Trp at the 28th position and the Tyr at the 599th position of the LAM were interaction point candidates, and the above two amino acids in Capra hircus LAM (chLAM), were successfully mutated to glycine by constructing recombinant yeast GS115/PIC9K-LAM M . The results showed that the sensitivity of Capra hircus LAM M (chLAM M ), to SW decreased significantly compared with wild-type LAM, the enzyme activity of LAM decreased approximately threefold, the optimum temperature of LAM M decreased from 55℃to 50℃, the optimum pH value increased from 4.5 to 5.0, and the effects of Mn 2+ , Fe 3+ , Al 3+ , Co 2+ , Cr 3+ , and Ethylene Diamine Tetraacetic Acid (EDTA) on LAM enzyme activity before and after point mutation changed significantly. These findings help us better understanding the molecular mechanism of the interaction mechanism between SW and chLAM, and provide new reference for solving locoweeds poisoning.

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Section: Discussion：mentioning

confidence: 99%

Biochemical characteristics of point mutated <i>Capra hircus</i> lysosome α-mannosidase

Wang

Zhang

Teng

et al. 2023

The Journal of Veterinary Medical Science

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“…Therefore, we can speculate that the participation of the SW molecule leads to the conformational change of the mannose ring, and the hydroxyl group at the 1 and 2 positions of the SW molecule plays an important role, preventing the hydrolysis of mannosidase. Compared with the parent, the β-mannanases (EC3.2.1.78) mutant screened by Zhang et al through random mutation has greatly improved speci c activity and thermal stability [34]; Another example is the mutation of four sites (43,44,55, and 62) with methionine on -amylase to alanine, the oxidative stability of the enzyme was signi cantly improved [35]. This further indicated that a slight conformational change appears to in uence the substrate-binding and enzyme catalytic characteristics [36].…”

Section: Discussionmentioning

confidence: 99%

Biochemical Characteristics of Point Mutated Goat Lysosome Α- Mannosidase

Yan

Zhang

Xiong

et al. 2022

Preprint

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Background Locoweeds are widely distributed all over the world. Goats and other herbivores often get poisoning if they eat a large number of locoweeds by mistake. Swainsonine (SW), the main toxin in locoweeds, can inhibit lysosomes α-Mannosidase (LAM) competitively in animal cells. The insufficient activity of lysosomes α-Mannosidase can lead to abnormal metabolism of animals, forming alpha mannosidosis. However，the details of interaction between SW and LAM are not clear yet. Methods In this study, molecular docking was used to predict the interaction points between SW and LAM. The effect of putative points was investigated by constructing mutated LAM (LAMM) and analyzing its biochemical characteristics. Results The results showed that the Trp at the 28th position and Tyr at the 599th position of the LAM were the candidates of interaction points. Both tryptophan at position 28 of A peptide residue and tyrosine at position 599 of D peptide residue of goat LAM were mutated into glycine, and goat lysosomes were obtained α- The mutant sequence of mannosidase; The recombinant yeast GS115 / pPIC9K LAMM was successfully constructed; After SW induction, the sensitivity of goat LAMM to SW decreased significantly, and the enzyme activity decreased about 3 times compared with wild-type LAM; The optimum temperature of LAMM decreased from 55 °C to 50 °C, and the optimum pH value increased from 4.5 to 5.0; All the trivalent metal ions (Fe3+, Al3+, Cr3+) had significant activation (P < 0.05), and EDTA had inhibitory effect on goat LAM before and after mutation. Conclusions These results indicated that the sensitivity of goat LAMM to SW was significantly reduced, but other characteristics changed little, which was consistent with the molecular docking results of goat LAMM. This study provides help for the study of interaction mechanism between SW and goat LAM, and also provides a new idea for the development of animal locoweed disease control technology.

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“…However, this study ( Ozturk et al, 2013 ) had proven that the mutation at near-to-active-site methionine residue (M55A) was more significant than mutation of solvent-accessible methionine residues (M43A + M44A) in terms of oxidative stability, where both types of methionine residues were proven to be oxidation prone ( Lin et al, 2003 ; Yang et al, 2012 ). This statement was justified when M55A mutant retained 50% of its initial activity in the presence of 100 mM H 2 O 2 , compared to double mutant (M43A + M44A) at 39% ( Ozturk et al, 2013 ). Nevertheless, such investigations and engineering to improve oxidative stability of microbial -amylases remain underexplored and should not become obsolete (latest report in 2013; Table 2 ) due to its great interests and advantages in detergent industry.…”

Section: Survey Methodologymentioning

confidence: 99%

Native to designed: microbial α-amylases for industrial applications

Lim

Oslan

2021

PeerJ

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Background α-amylases catalyze the endo-hydrolysis of α-1,4-D-glycosidic bonds in starch into smaller moieties. While industrial processes are usually performed at harsh conditions, α-amylases from mainly the bacteria, fungi and yeasts are preferred for their stabilities (thermal, pH and oxidative) and specificities (substrate and product). Microbial α-amylases can be purified and characterized for industrial applications. While exploring novel enzymes with these properties in the nature is time-costly, the advancements in protein engineering techniques including rational design, directed evolution and others have privileged their modifications to exhibit industrially ideal traits. However, the commentary on the strategies and preferably mutated residues are lacking, hindering the design of new mutants especially for enhanced substrate specificity and oxidative stability. Thus, our review ensures wider accessibility of the previously reported experimental findings to facilitate the future engineering work. Survey methodology and objectives A traditional review approach was taken to focus on the engineering of microbial α-amylases to enhance industrially favoured characteristics. The action mechanisms of α- and β-amylases were compared to avoid any bias in the research background. This review aimed to discuss the advances in modifying microbial α-amylases via protein engineering to achieve longer half-life in high temperature, improved resistance (acidic, alkaline and oxidative) and enhanced specificities (substrate and product). Captivating results were discussed in depth, including the extended half-life at 100 °C, pH 3.5 and 10, 1.8 M hydrogen peroxide as well as enhanced substrate (65.3%) and product (42.4%) specificities. These shed light to the future microbial α-amylase engineering in achieving paramount biochemical traits ameliorations to apt in the industries. Conclusions Microbial α-amylases can be tailored for specific industrial applications through protein engineering (rational design and directed evolution). While the critical mutation points are dependent on respective enzymes, formation of disulfide bridge between cysteine residues after mutations is crucial for elevated thermostability. Amino acids conversion to basic residues was reported for enhanced acidic resistance while hydrophobic interaction resulted from mutated hydrophobic residues in carbohydrate-binding module or surface-binding sites is pivotal for improved substrate specificity. Substitution of oxidation-prone methionine residues with non-polar residues increases the enzyme oxidative stability. Hence, this review provides conceptual advances for the future microbial α-amylases designs to exhibit industrially significant characteristics. However, more attention is needed to enhance substrate specificity and oxidative stability since they are least reported.

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Site-directed mutagenesis of methionine residues for improving the oxidative stability of α-amylase from Thermotoga maritima

Cited by 18 publications

References 13 publications

Biochemical characteristics of point mutated <i>Capra hircus</i> lysosome α-mannosidase

Biochemical characteristics of point mutated <i>Capra hircus</i> lysosome α-mannosidase

Biochemical Characteristics of Point Mutated Goat Lysosome Α- Mannosidase

Native to designed: microbial α-amylases for industrial applications

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