Correlation Analysis of Key Residue Sites between Computational-Aided Design Thermostability <scp>d</scp>-Amino Acid Oxidase and Ancestral Enzymes

Wang, Liu-Yu; Tang, Heng; Zhao, Jin-Qiao; Wang, Meng-Nan; Xue, Ya-Ping; Zheng, Yu-Guo

doi:10.1021/acs.jafc.3c06865

Cited by 3 publications

(2 citation statements)

References 36 publications

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“…Based on these findings, The residues were selected and located within 4.5 Å of either mogrol, mogroside IA1, or mogroside IIE for further analysis by docking (Figures c and S8). We also conducted activity-based sequence conservative analysis (ASCA) , of five previously reported UGTs from the UGT74 family to identify amino acid residues in the consensus flexible region that differ from those of UGT74DD1 (Figure S9). ASCA is based on the principle that amino acids present at high frequency at a specific position in homologous sequences have a greater impact on protein catalytic activity than those less frequently present at that position .…”

Section: Resultsmentioning

confidence: 99%

Rationally Engineered Novel Glycosyltransferase UGT74DD1 from Siraitia grosvenorii Catalyzes the Generation of the Sweetener Mogroside III

Gong,

Li,

Chu

et al. 2024

J. Agric. Food Chem.

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Mogrosides are natural compounds highly valued in the food sector for their exceptional sweetness. Here, we report a novel O-glycosyltransferase (UGT74DD1) from Siraitia grosvenorii that catalyzes the conversion of mogrol to mogroside IIE. Sitedirected mutagenesis yielded the UGT74DD1-W351A mutant, which exhibited the new capability to transform mogroside IIE into the valuable sweetener mogroside III, but with low catalytic activity. Subsequently, using structure-guided directed evolution with combinatorial active-site saturation testing, the superior mutant M6 (W351A/Q373 K/E49H/Q335W/S278C/D17F) were obtained, which showed a 46.1-fold increase in catalytic activity compared to UGT74DD1-W351A. Molecular dynamics simulations suggested that the enhanced activity and extended substrate profiles of M6 are due to its enlarged substrate-binding pocket and strengthened enzyme−substrate hydrogen bonding interactions. Overall, we redesigned UGT74DD1, yielding mutants that catalyze the conversion of mogrol into mogroside III. This study thus broadens the toolbox of UGTs capable of catalyzing the formation of valuable polyglycoside compounds.

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Section: Resultsmentioning

confidence: 99%

Rationally Engineered Novel Glycosyltransferase UGT74DD1 from Siraitia grosvenorii Catalyzes the Generation of the Sweetener Mogroside III

Gong,

Li,

Chu

et al. 2024

J. Agric. Food Chem.

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“…Electrostatic interactions also play an important role in protein stability, [16] and optimizing the distribution of electrostatic charges on protein surface can enhance thermostability. [35] The residue T232 was located close to a neutral region (Figure 3G), and substituting it with alanine created a larger neutral environment (Figure 3H), which stabilized the overall structure. [36] Our result is consistent with previ-ous studies, [36,37] which showed that thermophilic proteins have large hydrophobic surface areas and tightly packed hydrophobic layers.…”

Section: Mechanism Elucidation For Stability Enhancementmentioning

confidence: 99%

Computer‐aided design to enhance the stability of aldo‐keto reductase KdAKR

Dai,

Tian,

Chen

et al. 2024

Biotechnology Journal

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The aldo‐keto reductase (AKR) KdAKR from Kluyvermyces dobzhanskii can reduce t‐butyl 6‐chloro‐(5S)‐hydroxy‐3‐oxohexanoate ((5S)‐CHOH) to t‐butyl 6‐chloro‐(3R,5S)‐dihydroxyhexanoate ((3R,5S)‐CDHH), which is the key chiral intermediate of rosuvastatin. Herein, a computer‐aided design that combined the use of PROSS platform and consensus design was employed to improve the stability of a previously constructed mutant KdAKRM6. Experimental verification revealed that S196C, T232A, V264I and V45L produced improved thermostability and activity. The “best” mutant KdAKRM10 (KdAKRM6‐S196C/T232A/V264I/V45L) was constructed by combining the four beneficial mutations, which displayed enhanced thermostability. Its T5015 and Tm values were increased by 10.2 and 10.0°C, respectively, and half‐life (t1/2) at 40°C was increased by 17.6 h. Additionally, KdAKRM10 demonstrated improved resistance to organic solvents compared to that of KdAKRM6. Structural analysis revealed that the increased number of hydrogen bonds and stabilized hydrophobic core contributed to the rigidity of KdAKRM10, thus improving its stability. The results validated the feasibility of the computer‐aided design strategy in improving the stability of AKRs.

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Identification of efficient amine transaminase and applicability in dual transaminases cascade for synthesis of L-phosphinothricin

Yi,

Liu,

Hao

et al. 2024

Enzyme and Microbial Technology

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Correlation Analysis of Key Residue Sites between Computational-Aided Design Thermostability d-Amino Acid Oxidase and Ancestral Enzymes

Cited by 3 publications

References 36 publications

Rationally Engineered Novel Glycosyltransferase UGT74DD1 from Siraitia grosvenorii Catalyzes the Generation of the Sweetener Mogroside III

Rationally Engineered Novel Glycosyltransferase UGT74DD1 from Siraitia grosvenorii Catalyzes the Generation of the Sweetener Mogroside III

Computer‐aided design to enhance the stability of aldo‐keto reductase KdAKR

Identification of efficient amine transaminase and applicability in dual transaminases cascade for synthesis of L-phosphinothricin

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