- Reductase-catalyzed tetrahydrobiopterin regeneration alleviates the anti-competitive inhibition of tyrosine hydroxylation by 7,8-dihydrobiopterin
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l-Tyrosine hydroxylation by tyrosine hydroxylase is a significant reaction for preparing many nutraceutical and pharmaceutical chemicals. Two major challenges in constructing these pathways in bacteria are the improvement of hydroxylase catalytic efficiency and the production of cofactor tetrahydrobiopterin (BH4). In this study, we analyzed the evolutionary relationships and conserved protein sequences between tyrosine hydroxylases from different species by PhyML and MAFFT. Finally, we selected 7 tyrosine hydroxylases and 6 sepiapterin reductases. Subsequently, the function of different groups was identified by a combined whole-cell catalyst, and a series of novel tyrosine hydroxylase/sepiapterin reductase (TH/SPR) synthesis systems were screened including tyrosine hydroxylase (from Streptosporangium roseum DSM 43021 and Thermomonospora curvata DSM 43183) and sepiapterin reductase (from Photobacterium damselae, Chlorobaculum thiosulfatiphilum and Xenorhabdus poinarii), namely as SrTH/PdSPR, SrTH/CtSPR, SrTH/XpSPR and TcTH/PdSPR, which can synthesize l-Dopa by hydroxylating l-tyrosine in Bacillus licheniformis. Furthermore, we analyzed the characterization of SrTH by enzyme catalysis and demonstrated that 7,8-dihydrobiopterin (BH2) formed by BH4 autooxidation was an anticompetitive inhibitor on SrTH. Finally, pure dihydropteridine reductase from Escherichia coli (EcDHPR) was added to the solution, and l-Dopa could be continually synthesized after 3 h, which was improved by 86% at 6 h in the catalytic reaction by SrTH. This indicates that BH4 regeneration can alleviate the inhibition by BH2 during tyrosine hydroxylation. This study provides a good starting point and theoretical foundation for further modification to improve the catalytic efficiency of tyrosine hydroxylation by tyrosine hydroxylase.
- Ding, Zhongyang,Li, Leyun,Li, Youran,Shi, Guiyang,Xu, Yinbiao,Zhang, Liang
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p. 3128 - 3140
(2021/05/25)
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- METHOD OF SYNTHESIZING TETRAHYDROBIOPTERIN
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The present disclosure provides a method that efficiently produces (6R)-tetrahydrobiopterin in high yield and purity. The method includes the step of hydrolyzing diacetylbiopterin to biopterin under basic conditions in a biphasic mixture comprising an organic phase and an aqueous phase. After substantially complete hydrolysis of diacetylbiopterin, the aqueous phase containing biopterin can be separated from the organic phase containing most of the organic impurities, which avoids the time-consuming step of isolating biopterin as a solid. The aqueous solution containing biopterin is stereoselectively hydrogenated to (6R)-tetrahydrobiopterin under basic conditions and high hydrogen pressure in the presence of a metal catalyst (e.g., a platinum catalyst). To improve the purification of an acid addition salt of (6R)-tetrahydrobiopterin (e.g., (6R)-tetrahydrobiopterin dihydrochloride), any residual salts (e.g., sodium salts) in the aqueous solution after the hydrogenation reaction can be removed by contacting the aqueous solution with an ion (e.g., cation) exchange resin or column. Alternatively, removal of residual salts from the aqueous solution can be omitted if an organic amine (e.g., diethylamine or triethylamine) rather than an inorganic base is used in the hydrolysis and/or hydrogenation reactions.
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Page/Page column 11-12
(2009/08/16)
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- Quinonoid Dihydrobiopterin, an Important Metabolic Intermediate of Biopterin Cofactor in the Aromatic Hydroxylation of Amino Acids
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p-Quinonoid (6R)-dihydrobiopterin hydrochloride was synthesized from (6R)-5,6,7,8-tetrahydrobiopterin dihydrochloride by hydrogenperoxide in the presence of potassium iodide.Several characters of quinonoid dihydrobiopterin were examined.
- Matsuura, Sadao,Murata, Shizuaki,Sugimoto, Takashi
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p. 585 - 588
(2007/10/02)
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