Guangrong Sun,Pei Guo,Han Yeong Kaw,Meijiao Zhou,Wei Wang
Constant efforts have been devoted to exploring new disinfection byproducts in drinking water causally related to adverse health outcomes. In this study, five halogenated nucleobases were identified as emerging disinfection byproducts in drinking water, including 5-chlorouracil, 6-chlorouracil, 2-chloroadenine, 6-chloroguanine, and 5-bromouracil. We developed a solid phase extraction–ultraperformance liquid chromatography–tandem mass spectrometry method with the limits of detection (LOD) and recoveries ranging between 0.04–0.86 ng/L and 54–93%, respectively. The detection frequency of the five halogenated nucleobases ranged from 73 to 100% with a maximum concentration of up to 65.3 ng/L in the representative drinking water samples. The cytotoxicity of the five identified halogenated nucleobases in Chinese hamster ovary (CHO-K1) cells varied with great disparity, in which the cytotoxicity of 2-chloroadenine (IC50 = 9.4 μM) is appropriately three times higher than emerging DBP 2,6-dichloro-1,4-benzoquinone (IC50 = 42.4 μM), indicating the significant toxicological risk of halogenated nucleobase-DBPs. To the best of our knowledge, this study reports the analytical method, occurrence, and toxicity of halogenated nucleobase-DBPs for the first time. These findings will provide a theoretical basis for further research on probing the relationship between its mutagenicity and human health risk.
Multienzyme cascades provide an efficient biocatalytic strategy for synthesizing nucleoside analogs from simple sugar donors and nucleobases. In pentose‐to‐nucleoside cascades, purine nucleoside phosphorylase (PNP) catalyzes the terminal N‐glycosylation step and strongly influences product formation and substrate scope. Here, seven putative PNP homologs from microorganisms isolated from low‐temperature environments were evaluated under Ec RK‐ Ec PPM‐PNP cascade conditions. A PNP from Colwellia sp. WH041, designated Col PNP, showed the highest arabinofuranosylation activity and was selected for further characterization. Col PNP displayed optimal activity at 45 °C and exhibited a 12.7‐fold higher catalytic efficiency than Escherichia coli PNP at the same temperature. Molecular dynamics simulations supported the favorable catalytic performance of Col PNP under moderate‐temperature conditions. After optimization of the Ec RK‐ Ec PPM‐ Col PNP cascade, vidarabine conversion reached 58.2% within 12 h. The optimized system was further applied to synthesize diverse nucleoside analogs with conversions of 45.8%–89.8%, and guanosine analogs were obtained through sequential Ec ADA‐mediated deamination. This study expands the PNP toolbox and demonstrates Col PNP as an efficient module for enzymatic nucleoside analog synthesis. A purine nucleoside phosphorylase from Colwellia sp. WH041, Col PNP, is identified as an efficient module for pentose‐to‐purine nucleoside synthesis. Integrated with Ec RK and Ec PPM, Col PNP enables direct conversion of unprotected D‐pentoses and purine bases into diverse nucleoside analogs with up to 89.9% conversion.
Gaofei Liu,Xin Tong,Jialing Wang,Bin Wu,Jianlin Chu,Yongchan Jian,Bingfang He
2-Halogenated-2′-deoxyadenosines are important antiviral and anticancer drugs. However, natural enzymes display low activities in the biosynthesis of 2-halogenated-2′-deoxyadenosines, and the results are unsatisfactory because of the steric impediment created by the halogenated substituent group in purine. In this study, a semi-rational design was performed to enhance the activity of purine nucleoside phosphorylase from Aneurinibacillus migulanus AM007 (AmPNP) for the biosynthesis of 2-halogenated-2′-deoxyadenosines. Small and smart libraries of AmPNP were generated by stepwise site-directed evolution using iterative combinatorial mutations. The best AmPNP mutant, M3 (N233D/E191Q/Y190V/M249I), exhibited a satisfactory phosphorolysis activity towards 2′-deoxyadenosine, with a 144.75-fold improvement compared with that of wild-type AmPNP. In a coupled reaction of the M3 mutant and Brevibacillus borstelensis LK01 pyrimidine nucleoside phosphorylase, the conversion rates remarkably improved and reached 89.94% (cladribine) and 81.24% (2-fluoro-2′-deoxyadenosine) within 10 h, respectively. The substrate-binding cavity of the M3 mutant was reshaped to provide good accessibility for halogenated molecules, which led to a higher activity in the synthesis of 2-halogenated nucleosides. This directed evolution towards overcoming challenges in the biosynthesis of halogenated nucleosides may result in a remarkable economic impact on biotechnological nucleoside production.
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