A chemoenzymatic strategy for the efficient synthesis of amphenicol antibiotic chloramphenicol mediated by an engineered <scp>l</scp>-threonine transaldolase with high activity and stereoselectivity

Xu, Liang; Nie, Dan; Su, Bing-Mei; Xu, Xinqi; Lin, Juan

doi:10.1039/d2cy01670b

Cited by 6 publications

(3 citation statements)

References 29 publications

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“…In the transaldol reaction catalyzed by LTTAs, acetaldehyde is a simultaneous byproduct. ,,, Accumulation of excess acetaldehyde not only hampers enzyme activity but also inhibits the formation of desired products. , ADHs are among the most common enzymes used to eliminate acetaldehyde . They exhibit high catalytic activity and are frequently used for aldehyde reduction .…”

Section: Resultsmentioning

confidence: 99%

Deciphering the Key Loop: Enhancing l-Threonine Transaldolase’s Catalytic Potential

Xi,

Rao,

Zhang

et al. 2024

ACS Catal.

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L-Threonine transaldolase (LTTA) is an attractive biocatalyst because of its potential diastereoselectivity in the synthesis of β-hydroxy-αamino acids (βHAAs). However, prospective development of LTTA has been hampered by its low activity. Here, a combination of techniques involving structural comparison, computational analysis, Loop deletion, and alanine scanning was used to identify a key Loop region (Loop 1) regulating the catalytic ability of Chitiniphilus shinanonensis LTTA (CsLTTA). Saturation mutagenesis and iterative saturation mutagenesis at the hot spots in Loop 1 were performed, and the best variant containing an F70T/C57Q/Y69T (TQT) triple mutation was screened. The diastereoisomer excess (de) produced by the TQT variant (95.4% syn ) was greater than that produced by the wild-type (WT) enzyme (75.2% syn ), and the catalytic efficiency (k cat /K m ) of the TQT variant was four times higher than that of the wild-type enzyme. Molecular dynamics simulations, metadynamics simulations, and CAVER analysis revealed the critical role of the Loop 1 structure in regulating the hydrogen bond network and thus reshaping the active-site pocket to control the syn-tunnel direction. Further engineering of Loop 1 in ObiH, an LTTA responsible for obafluorin biosynthesis, resulted in the development of the F70T-C57Q-H69T (ObiH-TQT) variant producing a de of 97% syn . Using the ObiH-TQT variant for kilogram-scale synthesis of L-syn-pmethylsulfonylphenylserine, coupled with acetaldehyde elimination, resulted in space−time yields of up to 12.7 g L −1 h −1 . The method achieved 98.3% substrate conversion and 99.2% syn de within 6 h, marking the highest reported levels to date. The above findings will contribute to the industrial production of β-hydroxy-α-amino acids, offer insights into the mechanism of Loop regions regulating the catalytic function of LTTAs, and provide ideas for engineering other enzymes.

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Section: Resultsmentioning

confidence: 99%

Deciphering the Key Loop: Enhancing l-Threonine Transaldolase’s Catalytic Potential

Xi,

Rao,

Zhang

et al. 2024

ACS Catal.

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“…Serine hydroxymethyltransferases (SHMTs), threonine aldolases (TAs), and l -threonine transaldolases (LTTAs) have been reported for the asymmetric synthesis of βHAAs. − Among them, LTTAs have become a research hot spot because of their good stereoselectivity for the Cβ atom of products . − LTTAs belong to the aspartyl transferase fold-type I superfamily of pyridoxal phosphate (PLP)-dependent enzymes, which are found widely in various natural product synthesis pathways, such as for nucleoside antibiotics, , β-lactone antibiotics, , thioheptose nucleoside antibiotics, etc. FTase (4-Fluorothreonine transaldolase) was the first LTTA to be discovered; it functions in the synthesis of 4-fluorothreonine .…”

Section: Introductionmentioning

confidence: 99%

Rational Design of l-Threonine Transaldolase-Mediated System for Enhanced Florfenicol Intermediate Production

Xi,

Li,

Liu

et al. 2023

J. Agric. Food Chem.

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L-threo-p-methylsulfonylphenylserine (compound 1b) is the main intermediate of florfenicol, and its efficient synthesis has been the subject of current research. Herein, Burkholderia diffusa L-threonine transaldolase (BuLTTA) was rationally designed based on the sequence−structure−function relationship. A mutant M4 (Asn35Ser/Thr352Asn) could produce 35.5 mM 1b with 88.8% conversion and 93.8% diastereoselectivity, 314 and 129% of the values observed for wild-type BuLTTA. Molecular dynamics simulations indicated that the shortened distance between key active site residues and the transition state (PLP-1b) and the improved hydrogen bond force enhanced the catalytic performance of the M4 variant. Then, the mutant M4 was combined with K. kurtzmanii alcohol dehydrogenase (KkADH) to eliminate the BuLTTA-inhibiting byproduct acetaldehyde, and a cosubstrate was added to regenerate the ADH cofactor NADH. Under optimized conditions, the yield of 1b reached 115.2 mM with a conversion of 96% and a diastereoselectivity of 95.5%. This work provides a new strategy for the efficient and sustainable production of 1b.

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“…With the progress of protein engineering, it is possible to tailor enzymes with desired properties such as improved activity or enantioselectivity (Xu et al, 2023;Su et al, 2019). Semi-rational design is an e cient strategy for improved catalytic activities of enzymes by site-directed saturation mutagenesis of residues lining the catalytic center, which is suitable for engineering enzyme without accurate structure or mechanism (Xu et al, 2020;Pang et al, 2023;Wang et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

Efficient synthesis of 2’-deoxyguanosine in one-pot cascade by employing an engineered purine nucleoside phosphorylase from Brevibacterium acetylicum

Xu,

Li,

Lin

2023

World J Microbiol Biotechnol

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2'-deoxyguanosine is a key medical intermediate which could be applied for the synthesis of anti-cancer drug and biomarker in type 2 diabetes. In present study, an enzymatic cascade for the e cient synthesis of 2'-deoxyguanosine was proposed by employing thymidine phosphorylase from Escherichia coli (EcTP) and purine nucleoside phosphorylase from Brevibacterium acetylicum (BaPNP) in a one-pot whole cell catalysis. Semi-rational design of BaPNP was performed to enhance its activity, resulting a best triple variant BaPNP-Mu3 (E57A/T189S/L243I), with an overall 5.6-fold higher yield of 2'-deoxyguanosine as compared with BaPNP-Mu0. Molecular dynamics simulation revealed that the engineering of BaPNP-Mu3 led to a larger and more exible substrate entrance channel, which might contribute to its catalytic performance. Furthermore, by coordinating the expression of BaPNP-M3 and EcTP, a robust whole cell catalyst W05 was constructed, which could produce 14.8 mM 2'-deoxyguanosine with a high time-space yield (1.32 g/L/h) and therefore was very competitive for industrial applications.

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A chemoenzymatic strategy for the efficient synthesis of amphenicol antibiotic chloramphenicol mediated by an engineered l-threonine transaldolase with high activity and stereoselectivity

Abstract: Chloramphenicol, a kind of amphenicol antibiotic with broad-spectrum antibacterial activity, is challenging for synthesis due to its stereochemistry. Here we designed a four-step chemoenzymatic strategy, including a biocatalytic step mediated...

Cited by 6 publications

References 29 publications

Deciphering the Key Loop: Enhancing l-Threonine Transaldolase’s Catalytic Potential

Deciphering the Key Loop: Enhancing l-Threonine Transaldolase’s Catalytic Potential

Rational Design of l-Threonine Transaldolase-Mediated System for Enhanced Florfenicol Intermediate Production

Efficient synthesis of 2’-deoxyguanosine in one-pot cascade by employing an engineered purine nucleoside phosphorylase from Brevibacterium acetylicum

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