Biochemical characterization of a novel tyrosine phenol-lyase from Fusobacterium nucleatum for highly efficient biosynthesis of l-DOPA

Zheng, Ren‐Chao; Tang, Xiaoling; Suo, Hui; Feng, Lilin; Liu, Xiao; Yang, Jian; Zheng, Yu‐Guo

doi:10.1016/j.enzmictec.2017.11.004

Cited by 36 publications

(19 citation statements)

References 23 publications

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“…By detecting the final reaction mixture, it was found that the remaining pyruvate concentration was 15.3 % lower in the one‐step system than that in the two‐step coupled cascade reaction (Figure S8). As high donor/acceptor ratio can facilitate the C‐C coupling due to the low affinity of most TPL towards pyruvate,[4a], the cascade probably enhanced the yield of L‐4f by avoiding the decrease of pyruvate as well as donor/acceptor ratio. According to previous studies, pyruvate is not stable and can undergo an aldol‐type condensation to form polymers, especially under alkaline conditions at high concentrations …”

Section: Resultsmentioning

confidence: 99%

“…The classical chemical synthesis of these tyrosine derivatives usually comprises several steps including protection and deprotection of the functional amino acid groups and leads to racemic material, which requires late‐stage racemate resolution . In contrast, PLP‐dependent tyrosine‐phenol lyases (TPL, EC 4.1.99.2) can catalyze the regio‐ and stereoselective reversible formation of tyrosine derivatives from donor substrate pyruvate and acceptor substrate phenol or derivatives in a single and green reaction . Among various TPLs, thermophilic TPL from Symbiobacterium toebii (TTPL) exhibited markedly high stability against phenols and have been employed as biocatalysts for enzymatic synthesis of l ‐tyrosine and L‐DOPA .…”

Section: Introductionmentioning

confidence: 99%

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Biocascade Synthesis of L‐Tyrosine Derivatives by Coupling a Thermophilic Tyrosine Phenol‐Lyase and L‐Lactate Oxidase

Lian

Xue

et al. 2020

Eur J Org Chem

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A one‐pot biocascade of two enzymatic steps catalyzed by an l‐lactate oxidase and a tyrosine phenol‐lyase has been successfully developed in the present study. The reaction provides an efficient method for the synthesis of l‐tyrosine derivatives, which exhibits readily available starting materials and excellent yields. In the first step, an in situ generation of pyruvate from readily available bio‐based l‐lactate catalyzed by a highly active l‐lactate oxidase from Aerococcus viridans (AvLOX) was developed (using oxygen as oxidant and catalase as hydrogen peroxide removing reagent). Pyruvate thus produced underwent C–C coupling with phenol derivatives as acceptor substrate using specially designed thermophilic tyrosine phenol‐lyase mutants from Symbiobacterium toebii (TTPL). Overall, this cascade avoids the high cost and easy decomposition of pyruvate and offered an efficient and environmentally friendly procedure for l‐tyrosine derivatives synthesis.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Biocascade Synthesis of L‐Tyrosine Derivatives by Coupling a Thermophilic Tyrosine Phenol‐Lyase and L‐Lactate Oxidase

Lian

Xue

et al. 2020

Eur J Org Chem

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“…The optimal temperature was estimated in 50 mM Tris-HCl buffer (pH 8.5) with 10 mM L -valine, 4 mM α-ketoglutaric acid and 10 μ M PLP under different temperature (25~55 °C). The effect of PLP concentration on activity was tested at 37 °C by performing 10 mM L -valine and 4 mM α-ketoglutaric acid reactions in 50 mM Tris-HCl buffer (pH 8.5) (22).…”

Section: Methodsmentioning

confidence: 99%

Biochemical and structural characterization of a highly active branched-chain amino acid aminotransferase from Pseudomonas sp. for efficient biosynthesis of chiral amino acids

Zheng

Cui

et al. 2019

Preprint

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Aminotransferases (ATs) are important biocatalysts for the synthesis of chiral amines because of their capability of introducing amino group into ketones or keto acids as well as their high enantioselectivity, high regioselectivity and no requirement of external addition of cofactor. Among all ATs, branched-chain amino acid aminotransferase (BCAT) can reversibly catalyse branched-chain amino acids (BCAAs), including L-valine, L-leucine, and L-isoleucine, with α-ketoglutaric acid to form the corresponding ketonic acids and L-glutamic acid. Alternatively, BCATs have been used for the biosynthesis of unnatural amino acids, such as L-tert-leucine. In the present study, the BCAT from Pseudomonas sp. (PsBCAT) was cloned and expressed in Escherichia coli for biochemical and structural analyses. The optimal reaction temperature and pH of PsBCAT were 40 °C and 8.5, respectively. PsBCAT exhibited a comparatively broader substrate spectrum, and showed remarkably high activity with L-leucine, L-valine, L-isoleucine and L-methionine with activities of 105 U/mg, 127 U/mg, 115 U/mg and 98 U/mg, respectively. Additionally, PsBCAT had activities with aromatic L-amino acids, L-histidine, L-lysine, and L-threonine. To analyse the catalytic mechanism of PsBCAT with the broad substrate spectrum, the crystal structure of PsBCAT was also determined. Finally, conjugated with the ornithine aminotransferase (OrnAT) from Bacillus subtilis, the coupled system was applied to the preparation of L-tert-leucine with 83% conversion, which provided an approximately 2.7-fold higher yield than the single BCAT reaction.IMPORTANCEDespite the enormous potential of BCATs, the vast majority of enzymes still lack suitably broad substrate scope and activity, thus new sources and novel enzymes are currently being investigated. Here, we described a previously uncharacterized PsBCAT, which showed a surprisingly wide substrate range and was more active towards BCAAs. This substrate promiscuity is unique for the BCAT family and could prove useful in industrial applications. Based on the determined crystal structure, we found some differences in the organization of the substrate binding cavity, which may influence the substrate specificity of the enzyme. Moreover, we demonstrated efficient biocatalytic asymmetric synthesis of L-tert-leucine using a coupling system, which can be used to remove the inhibitory by-product, and to shift the reaction equilibrium towards the product formation. In summary, the structural and functional characteristics of PsBCAT were analysed in detail, and this information will play an important role in the synthesis of chiral amino acids and will be conducive to industrial production of enantiopure chiral amines by aminotransferase.

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“…TPL does not belong to enzyme with good thermostability. Therefore, reaction catalyzed by TPL was generally performed at 15 °C [62]. The maximal enzyme activity of TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16 was 5.0 IU/mL, 5.4 IU/mL, 4.7 IU/mL, and 3.9 IU/mL, respectively, exhibiting improvement by 117.4%, 133.0%, 104.3%, and 69.5% relative to TPL at 20 °C.…”

Section: Catalytic Properties Of Tplsmentioning

confidence: 99%

“…Tyrosine phenol-lyase (TPL) (EC4.1.99.2), a tetrameric enzyme, can catalyze stereospecific isotope exchange of α -protons of various amino acids with pyridoxal-5′-phosphate (PLP) as the cofactor [ 20 ]. TPL has been mainly isolated and characterized from bacteria, such as Citrobacter freundii [ 38 , 46 ], Erwinia herbicola [ 58 ], and Fusobacterium nucleatum [ 62 ]. Monovalent cations, K + or NH 4 + are necessary for achieving high activity levels of TPL [ 33 ].…”

Section: Introductionmentioning

confidence: 99%

Active tyrosine phenol-lyase aggregates induced by terminally attached functional peptides in Escherichia coli

Han

Zeng

Zhang

et al. 2020

Journal of Industrial Microbiology and Biotechnology

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The formation of inclusion bodies (IBs) without enzyme activity in bacterial research is generally undesirable. Researchers have attempted to recovery the enzyme activities of IBs, which are commonly known as active IBs. Tyrosine phenol-lyase (TPL) is an important enzyme that can convert pyruvate and phenol into 3,4-dihydroxyphenyl-l-alanine (L-DOPA) and IBs of TPL can commonly occur. To induce the correct folding and recover the enzyme activity of the IBs, peptides, such as ELK16, DKL6, L6KD, ELP10, ELP20, L6K2, EAK16, 18A, and GFIL16, were fused to the carboxyl terminus of TPL. The results showed that aggregate particles of TPL-DKL6, TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16 improved the enzyme activity by 40.9%, 50.7%, 48.9%, 86.6%, and 97.9%, respectively. The peptides TPL-DKL6, TPL-EAK16, TPL-18A, and TPL-GFIL16 displayed significantly improved thermostability compared with TPL. L-DOPA titer of TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16, with cells reaching 37.8 g/L, 53.8 g/L, 37.5 g/L, and 29.1 g/L, had an improvement of 111%, 201%, 109%, and 63%, respectively. A higher activity and L-DOPA titer of the TPL-EAK16 could be valuable for its industrial application to biosynthesize L-DOPA.

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Biochemical characterization of a novel tyrosine phenol-lyase from Fusobacterium nucleatum for highly efficient biosynthesis of l-DOPA

Cited by 36 publications

References 23 publications

Biocascade Synthesis of L‐Tyrosine Derivatives by Coupling a Thermophilic Tyrosine Phenol‐Lyase and L‐Lactate Oxidase

Biocascade Synthesis of L‐Tyrosine Derivatives by Coupling a Thermophilic Tyrosine Phenol‐Lyase and L‐Lactate Oxidase

Biochemical and structural characterization of a highly active branched-chain amino acid aminotransferase from Pseudomonas sp. for efficient biosynthesis of chiral amino acids

Active tyrosine phenol-lyase aggregates induced by terminally attached functional peptides in Escherichia coli

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