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Báo cáo khoa học: Inactivation of tyrosine phenol-lyase by Pictet–Spengler reaction and alleviation by T15A mutation on intertwined N-terminal arm

Citrobacter freundiil-tyrosine phenol-lyase (TPL) was inactivated by a Pictet–Spengler reaction between the cofactor and a substrate, 3,4-dihyd-roxyphenyl-l-alanine (l-dopa), in proportion to an increase in the reaction temperature. Random mutagenesis of the tpl gene resulted in the genera-tion of a Thr15 to Ala mutant (T15A), which exhibited a two-fold improved activity towards l-DOPA as the substrate. | ễFEBS Journal Inactivation of tyrosine phenol-lyase by Pictet-Spengler reaction and alleviation by T15A mutation on intertwined N-terminal arm Seung-Goo Lee1 Seung-Pyo Hong2 Do Young Kim1 Jae Jun Song1 Hyeon-Su Ro3 and Moon-Hee Sung2 4 1 Systems Microbiology Research Center KRIBB Daejeon Korea 2 Bioleaders Corporation Daejeon Korea 3 Department of Microbiology and Research Institute of Life Science KyeongSang NationalUniversity Chinju Korea 4 Department of Bio- and Nanochemistry Kookmin University Seoul Korea Keywords cofactor affinity L-DOPA N-terminalarm Pictet-Spengler condensation tyrosine phenol-lyase Correspondence M.-H. Sung Department of Bio- and Nanochemistry Kookmin University Seoul136-702 Korea Fax 82 2 910 4415 Tel 82 2 910 4808 5098 E-mail smoonhee@kookmin.ac.kr Received 27 August 2006 revised 16 October 2006 accepted 18 October 2006 doi 10.1111 j.1742-4658.2006.05546.x Citrobacter freundii L-tyrosine phenol-lyase TPL was inactivated by a Pictet-Spengler reaction between the cofactor and a substrate 3 4-dihyd-roxyphenyl-L-alanine l-dopa in proportion to an increase in the reaction temperature. Random mutagenesis of the tpl gene resulted in the generation of a Thr15 to Ala mutant T15A which exhibited a two-fold improved activity towards L-DOPA as the substrate. The Thr15 residue was located on the intertwined N-terminal arm of the TPL structure and comprised an H-bond network in proximity to the hydrophobic core between the catalytic dimers. The maximum activity of the mutant and native enzymes with L-DOPA was detected at 45 and 40 C respectively which was 15 C lower than when using L-tyrosine as the substrate. The half-lives at 45 C were about 16.8 and 6.4 min for the mutant and native enzymes respectively in 10 mM L-DOPA. On treatment with excess pyrid-oxal-5 -phosphate PLP the L-DOPA-inactivated enzymes recovered over 80 of their original activities thereby attributing the inactivation to a loss of the cofactor through Pictet-Spengler condensation .