Engineering a thermostable version of D-allulose 3-epimerase from Rhodopirellula baltica via site-directed mutagenesis based on B-factors analysis

Mao, Shuhong; Cheng, Xiaotao; Zhu, Zhangliang; Chen, Ying; Li, Chao; Zhu, Mingjian; Liu, Xin; Lu, Fuping; Qin, Hao

doi:10.1016/j.enzmictec.2019.109441

Cited by 41 publications

(16 citation statements)

References 61 publications

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“…[23], Dorea sp. [24], Rhodopirellula baltica [25], Ruminococcus sp. [26], Staphylococcus aureus [27] and Treponema primitia [28], and ketose 3-epimerase from Arthrobacter globiformis [29].…”

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confidence: 99%

Crystal structure of a novel homodimeric l‐ribulose 3‐epimerase from Methylomonus sp.

et al. 2021

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D-Allulose has potential as a low-calorie sweetener which can suppress fat accumulation. Several enzymes capable of D-allulose production have been isolated, including D-tagatose 3-epimerases. Here, we report the isolation of a novel protein from Methylomonas sp. expected to be a putative enzyme based on sequence similarity to ketose 3-epimerase. The synthesized gene encoding the deduced ketose 3-epimerase was expressed as a recombinant enzyme in Escherichia coli, and it exhibited the highest enzymatic activity toward L-ribulose, followed by D-ribulose and D-allulose. The X-ray structure analysis of L-ribulose 3-epimerase from Methylomonas sp. (MetLRE) revealed a homodimeric enzyme, the first reported structure of dimeric Lribulose 3-epimerase. The monomeric structure of MetLRE is similar to that of homotetrameric L-ribulose 3-epimerases, but the short C-terminal a-helix of MetLRE is unique and different from those of known L-ribulose 3 epimerases. The length of the C-terminal a-helix was thought to be involved in tetramerization and increasing stability; however, the addition of residues to MetLRE at the C terminus did not lead to tetramer formation. MetLRE is the first dimeric L-ribulose 3-epimerase identified to exhibit high relative activity toward D-allulose. D-Allulose (alternative name D-psicose) is a rare sugar, and its physiological functions, such as altering the blood glucose level, suppressing fat accumulation, and use as a low-calorie sweetener [1-9], have been focused on as a promising functional food ingredient. In Japan, a low-calorie syrup containing D-allulose, Rare Sugar Sweet Ò (Matsutani Chemical Industry Co., Ltd., Hyogo, Japan) [10], is commercially available and its labeling as a functional food was recently approved. In addition, the U.S. Food and Drug Administration (FDA) stated that 'it allows the low-calorie sweetener allulose to be excluded from total and added sugar counts on Nutrition and Supplement Facts labels when used as an ingredient' (FDA In Brief, April, 2019). AbbreviationsAgDAE, D-allulose 3-epimerase from Arthrobacter globiformis; AtDAE, D-allulose 3-epimerase from Agrobacterium tumefaciens; CcDAE, D-allulose 3-epimerase from Clostridium cellulolyticum; DAE, D-allulose 3-epimerase; his_MetLRE long2, his_MetLRE with nine additional residues (TAIKTIELH) at the C terminus and with three mutations (Y9I, Y37F, Y286L); his_MetLRE, N-terminal his-tagged MetLRE; his_MetLRE3, his_MetLRE with three mutations (Y9I, Y37F, and Y286L); LRE, L-ribulose 3-epimerase; MetLRE, L-ribulose 3-epimerase from Methylomonas sp.; MlLRE, L-ribulose 3-epimerase from Mesorhizobium loti; PcDTE, D-tagatose 3-epimerases from Pseudomonas cichorii.

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confidence: 99%

Crystal structure of a novel homodimeric l‐ribulose 3‐epimerase from Methylomonus sp.

et al. 2021

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“…To decrease the number of variants that need to be screened experimentally, there are methods available to prioritize substitutions by predicting the early unfolding region of a protein and only mutate at positions with the highest priority. The B‐fitter approach [56–60] is based on the idea that highly flexible parts of the protein are more likely to be the first sites of unfolding. While the usefulness of such methods is often reported and stabilized enzymes are indeed obtained, hardly ever controls are presented in which also substitutions at positions that are predicted to be unimportant, are characterized.…”

Section: Discussionmentioning

confidence: 99%

Stabilizing AqdC, a Pseudomonas Quinolone Signal‐Cleaving Dioxygenase from Mycobacteria, by FRESCO‐Based Protein Engineering

et al. 2020

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The mycobacterial PQS dioxygenase AqdC, a cofactor‐less protein with an α/β‐hydrolase fold, inactivates the virulence‐associated quorum‐sensing signal molecule 2‐heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) produced by the opportunistic pathogen Pseudomonas aeruginosa and is therefore a potential anti‐virulence tool. We have used computational library design to predict stabilizing amino acid replacements in AqdC. While 57 out of 91 tested single substitutions throughout the protein led to stabilization, as judged by increases in Tappm of >2 °C, they all impaired catalytic activity. Combining substitutions, the proteins AqdC‐G40K‐A134L‐G220D‐Y238W and AqdC‐G40K‐G220D‐Y238W showed extended half‐lives and the best trade‐off between stability and activity, with increases in Tappm of 11.8 and 6.1 °C and relative activities of 22 and 72 %, respectively, compared to AqdC. Molecular dynamics simulations and principal component analysis suggested that stabilized proteins are less flexible than AqdC, and the loss of catalytic activity likely correlates with an inability to effectively open the entrance to the active site.

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“…The change of amino acid residues affects the advanced structure of DTEase, thus leading to the change of enzymatic properties. Sitedirected mutagenesis was used to enhance the thermostability of DAEase from R. baltica (Mao et al, 2020) and improve the catalytic behavior of C. obsidiansis L-rhamnose isomerase (Chen et al, 2018). The improvement of DTEase family enzymes and production processing will decrease the production cost and D-allulose price, thus ensuring that D-allulose will be conveniently available to consumers.…”

Section: Prospectsmentioning

confidence: 99%

Review on D-Allulose: In vivo Metabolism, Catalytic Mechanism, Engineering Strain Construction, Bio-Production Technology

Jiang

Xiao

Zhu

et al. 2020

Front. Bioeng. Biotechnol.

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Rare sugar D-allulose as a substitute sweetener is produced through the isomerization of D-fructose by D-tagatose 3-epimerases (DTEases) or D-allulose 3-epimerases (DAEases). D-Allulose is a kind of low energy monosaccharide sugar naturally existing in some fruits in very small quantities. D-Allulose not only possesses high value as a food ingredient and dietary supplement, but also exhibits a variety of physiological functions serving as improving insulin resistance, antioxidant enhancement, and hypoglycemic controls, and so forth. Thus, D-allulose has an important development value as an alternative to high-energy sugars. This review provided a systematic analysis of D-allulose characters, application, enzymatic characteristics and molecular modification, engineered strain construction, and processing technologies. The existing problems and its proposed solutions for D-allulose production are also discussed. More importantly, a green and recycling process technology for D-allulose production is proposed for low waste formation, low energy consumption, and high sugar yield.

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Engineering a thermostable version of D-allulose 3-epimerase from Rhodopirellula baltica via site-directed mutagenesis based on B-factors analysis

Cited by 41 publications

References 61 publications

Crystal structure of a novel homodimeric l‐ribulose 3‐epimerase from Methylomonus sp.

Crystal structure of a novel homodimeric l‐ribulose 3‐epimerase from Methylomonus sp.

Stabilizing AqdC, a Pseudomonas Quinolone Signal‐Cleaving Dioxygenase from Mycobacteria, by FRESCO‐Based Protein Engineering

Review on D-Allulose: In vivo Metabolism, Catalytic Mechanism, Engineering Strain Construction, Bio-Production Technology

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