3270-98-2Relevant articles and documents
High-throughput assay of tyrosine phenol-lyase activity using a cascade of enzymatic reactions
Zhu, Hang-Qin,Hu, Wen-Ye,Tang, Xiao-Ling,Zheng, Ren-Chao,Zheng, Yu-Guo
, (2022/01/19)
Tyrosine phenol-lyase (TPL) exhibits great potential in industrial biosynthesis of L-tyrosine and its derivates. To uncover and screen TPLs with excellent catalytic properties, there is unmet demand for development of facile and reliable screening system for TPL. Here we presented a novel assay format for the detection of TPL activity based on catechol 2,3-dioxygenase (C23O)-catalyzed reaction. Catechol released from TPL-catalyzed cleavage of 3,4-dihydroxy-L-phenylalanine (L-DOPA) was further oxidized by C23O to form 2-hydroxymuconate semialdehyde, which could be readily detected by spectrophotometric measurements at 375 nm. The assay achieved a unique balance between the ease of operation and superiority of analytical performances including linearity, sensitivity and accuracy. In addition, this assay enabled real-time monitoring of TPL activity with high efficiency and reliability. As C23O is highly specific towards catechol, a non-natural product of microorganism, the assay was therefore accessible to both crude cell extracts and the whole-cell system without elaborate purification steps of enzymes, which could greatly expedite discovery and engineering of TPLs. This study provided fundamental principle for high-throughput screening of other enzymes consuming or producing catechol derivatives.
Expression, purification, characterization and in silico analysis of newly isolated hydrocarbon degrading bleomycin resistance dioxygenase
Sharma, Vinay,Kumar, Rajender,Sharma, Vishal Kumar,Yadav, Ashok kumar,Tiirola, Marja,Sharma, Pushpender Kumar
, p. 533 - 544 (2019/11/19)
In the present investigation, we report cloning, expression, purification and characterization of a novel Bleomycin Resistance Dioxygenase (BRPD). His-tagged fusion protein was purified to homogeneity using Ni-NTA affinity chromatography, yielding 1.2 mg of BRPD with specific activity of 6.25 U mg?1 from 600 ml of E. coli culture. Purified enzyme was a dimer with molecular weight ~ 26 kDa in SDS-PAGE and ~ 73 kDa in native PAGE analysis. The protein catalyzed breakdown of hydrocarbon substrates, including catechol and hydroquinone, in the presence of metal ions, as characterized via spectrophotometric analysis of the enzymatic reactions. Bleomycin binding was proven using the EMSA gel retardation assay, and the putative bleomycin binding site was further determined by in silico analysis. Molecular dynamic simulations revealed that BRPD attains octahedral configuration in the presence of Fe2+ ion, forming six co-ordinate complexes to degrade hydroquinone-like molecules. In contrary, in the presence of Zn2+ ion BRPD adopts tetrahedral configuration, which enables degradation of catechol-like molecules.
Single Turnover Reveals Oxygenated Intermediates in Toluene/o-Xylene Monooxygenase in the Presence of the Native Redox Partners
Liang, Alexandria Deliz,Lippard, Stephen J.
supporting information, p. 10520 - 10523 (2015/09/28)
Toluene/o-xylene monooxygenase (ToMO) is a non-heme diiron protein that activates O2 for subsequent arene oxidation. ToMO utilizes four protein components, a catalytic hydroxylase, a regulatory protein, a Rieske protein, and a reductase. O2 activation and substrate hydroxylation in the presence of all four protein components is examined. These studies demonstrate the importance of native reductants by revealing reactivity unobserved when dithionite and mediators are used as the reductant. This reactivity is compared with that of other O2-activating diiron enzymes.
