Altering the substrate specificity of recombinant l-rhamnose isomerase from Thermoanaerobacterium saccharolyticum NTOU1 to favor d-allose production

Tseng, Wen‐Chi; Chen, Yu‐Chun; Chang, Hao-Chin; Lin, Chwen-Yea; Fang, Tsuei‐Yun

doi:10.1016/j.jbiotec.2022.08.015

Cited by 8 publications

(6 citation statements)

References 33 publications

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“…RMSD is negatively correlated with protein stability, with mutant variants exhibiting lower RMSD values and higher thermal stability. 25 Therefore, this newly formed hydrogen bond likely led to tighter binding between the active pocket and the substrate, 26 increasing its substrate affinity and thereby enhancing both catalytic activity and thermal stability. Analysis of the molecular structures of enzymes showed that simultaneously enhancing the thermal stability and catalytic Hence, the double mutant exhibited an increase in optimal temperature as well as enhanced thermal stability.…”

Section: ■ Discussionmentioning

confidence: 99%

“…Hydrogen bonding is one of the main types of interactions determining the strength of enzyme–substrate binding . More hydrogen bonds formed between the substrate and the catalytic residues and/or shorter distances to certain important residues result in tighter connections between the substrate and the active site, thus enhancing the catalytic activity of the enzyme …”

Section: Discussionmentioning

confidence: 99%

“…It is probable that hydrogen bonds were the primary factor underpinning changes in the rigidity change in the active site. RMSD is negatively correlated with protein stability, with mutant variants exhibiting lower RMSD values and higher thermal stability . Therefore, this newly formed hydrogen bond likely led to tighter binding between the active pocket and the substrate, increasing its substrate affinity and thereby enhancing both catalytic activity and thermal stability.…”

Section: Discussionmentioning

confidence: 99%

“…20 More hydrogen bonds formed between the substrate and the catalytic residues and/or shorter distances to certain important residues result in tighter connections between the substrate and the active site, thus enhancing the catalytic activity of the enzyme. 25 The behavior of the double mutant is consistent with Cb-I265 V; a new hydrogen bond is formed with residue 162 that allows tighter binding of the substrate in the active pocket, increasing its substrate affinity. The increase in the number of hydrogen bonds significantly decreased the RMSF value of amino acids near residue 256, a region housing the active site.…”

Section: ■ Discussionmentioning

confidence: 99%

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Simultaneous Enhancement of the Thermostability and Catalytic Activity of D-Allulose 3-Epimerase from Clostridium bolteae ATTC BAA-613 Based on the “Back to Consensus Mutations” Hypothesis

Wang,

Feng

et al. 2024

J. Agric. Food Chem.

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D-Allulose is a high value rare sugar with multiple physiological functions and commercial potential that can be enzymatically synthesized from D-fructose by D-allulose 3-epimerase (DAEase). Poor catalytic activity and thermostability of DAEase prevent the industrial production of D-allulose. In this work, rational design was applied to a previously identified DAEase from Clostridium bolteae ATCC BAA-613 based on the "back to consensus mutations" hypothesis, and the catalytic activity of the Cb-I265 V variant was enhanced 2.5-fold. Furthermore, the Cb-I265 V/E268D double-site variant displayed 2.0-fold higher specific catalytic activity and 1.4-fold higher thermostability than the wild-type enzyme. Molecular docking and kinetic simulation results indicated increased hydrogen bonds between the active pocket and substrate, possibly contributing to the improved thermal stability and catalytic activity of the double-site mutant. The findings outlined a feasible approach for the rational design of multiple preset functions of target enzymes simultaneously.

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Section: ■ Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: ■ Discussionmentioning

confidence: 99%

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Simultaneous Enhancement of the Thermostability and Catalytic Activity of D-Allulose 3-Epimerase from Clostridium bolteae ATTC BAA-613 Based on the “Back to Consensus Mutations” Hypothesis

Wang,

Feng

et al. 2024

J. Agric. Food Chem.

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“…The results of activity screening against different substrates showed that I102N, I102Q, and I102R variants significantly increased the substrate preference towards D-allose with the catalytic efficiencies being 148%, 277% and 191% respectively, compared to the wild-type. The increased activity of I102Q could be attributed to the introduction of a hydrogen bond, which stabilized the enzyme-allose complex and increased the substrate affinity to the enzyme ( Tseng et al, 2022 ). In addition, a rational design strategy based on remodeling the hydrophobic interactions has also been applied to improve the acyltransferase activity of an esterase from Pyrobaculum calidifontis VA1 (PestE).…”

Section: Rational Design Strategy Based On Remodeling Interaction Net...mentioning

confidence: 99%

Rational design of enzyme activity and enantioselectivity

Song

Zhang

Wu³

et al. 2023

Front. Bioeng. Biotechnol.

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The strategy of rational design to engineer enzymes is to predict the potential mutants based on the understanding of the relationships between protein structure and function, and subsequently introduce the mutations using the site-directed mutagenesis. Rational design methods are universal, relatively fast and have the potential to be developed into algorithms that can quantitatively predict the performance of the designed sequences. Compared to the protein stability, it was more challenging to design an enzyme with improved activity or selectivity, due to the complexity of enzyme molecular structure and inadequate understanding of the relationships between enzyme structures and functions. However, with the development of computational force, advanced algorithm and a deeper understanding of enzyme catalytic mechanisms, rational design could significantly simplify the process of engineering enzyme functions and the number of studies applying rational design strategy has been increasing. Here, we reviewed the recent advances of applying the rational design strategy to engineer enzyme functions including activity and enantioselectivity. Five strategies including multiple sequence alignment, strategy based on steric hindrance, strategy based on remodeling interaction network, strategy based on dynamics modification and computational protein design are discussed and the successful cases using these strategies are introduced.

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X-ray structure and characterization of a probiotic Lactobacillus rhamnosus Probio-M9 L-rhamnose isomerase

Yoshida,

Yamamoto,

Kurahara

et al. 2024

Appl Microbiol Biotechnol

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A recombinant L-rhamnose isomerase (L-RhI) from probiotic Lactobacillus rhamnosus Probio-M9 (L. rhamnosus Probio-M9) was expressed. L. rhamnosus Probio-M9 was isolated from human colostrum and identified as a probiotic lactic acid bacterium, which can grow using L-rhamnose. L-RhI is one of the enzymes involved in L-rhamnose metabolism and catalyzes the reversible isomerization between L-rhamnose and L-rhamnulose. Some L-RhIs were reported to catalyze isomerization not only between L-rhamnose and L-rhamnulose but also between D-allulose and D-allose, which are known as rare sugars. Those L-RhIs are attractive enzymes for rare sugar production and have the potential to be further improved by enzyme engineering; however, the known crystal structures of L-RhIs recognizing rare sugars are limited. In addition, the optimum pH levels of most reported L-RhIs are basic rather than neutral, and such a basic condition causes non-enzymatic aldose-ketose isomerization, resulting in unexpected by-products. Herein, we report the crystal structures of L. rhamnosus Probio-M9 L-RhI (LrL-RhI) in complexes with L-rhamnose, D-allulose, and D-allose, which show enzyme activity toward L-rhamnose, D-allulose, and D-allose in acidic conditions, though the activity toward D-allose was low. In the complex with L-rhamnose, L-rhamnopyranose was found in the catalytic site, showing favorable recognition for catalysis. In the complex with D-allulose, D-allulofuranose and ring-opened D-allulose were observed in the catalytic site. However, bound D-allose in the pyranose form was found in the catalytic site of the complex with D-allose, which was unfavorable for recognition, like an inhibition mode. The structure of the complex may explain the low activity toward D-allose. Key points • Crystal structures of LrL-RhI in complexes with substrates were determined. • LrL-RhI exhibits enzyme activity toward L-rhamnose, D-allulose, and D-allose. • The LrL-RhI is active in acidic conditions.

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Altering the substrate specificity of recombinant l-rhamnose isomerase from Thermoanaerobacterium saccharolyticum NTOU1 to favor d-allose production

Cited by 8 publications

References 33 publications

Simultaneous Enhancement of the Thermostability and Catalytic Activity of D-Allulose 3-Epimerase from Clostridium bolteae ATTC BAA-613 Based on the “Back to Consensus Mutations” Hypothesis

Simultaneous Enhancement of the Thermostability and Catalytic Activity of D-Allulose 3-Epimerase from Clostridium bolteae ATTC BAA-613 Based on the “Back to Consensus Mutations” Hypothesis

Rational design of enzyme activity and enantioselectivity

X-ray structure and characterization of a probiotic Lactobacillus rhamnosus Probio-M9 L-rhamnose isomerase

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