Expression and functional characterization of prephenate dehydrogenase from Streptococcus mutans

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• 作者：Hyung-Keun Ku ; Sang-Ryoul Park ; Inchul Yang ; Sook-Kyung Kim
• 关键词：Prephenate dehydrogenase ; Streptococcus mutans ; MALDI-TOF ; Size exclusion chromatography ; Site-directed mutagenesis ; Tyrosine
• 刊名：Process Biochemistry
• 出版年：2010
• 出版时间：April 2010
• 年：2010
• 卷：45
• 期：4
• 页码：607-612
• 全文大小：572 K

文摘

Streptococcus mutans is commonly found in the human oral cavity. Functionally active prephenate dehydrogenase (PDH) catalyzes conversion of prephenate to 4-hydroxylphenylpyruvate. In order to characterize S. mutans-PDH (Sm-PDH), a pdh gene was expressed using Escherichia coli expression vector pET-15b and Sm-PDH was affinity purified using a nickel column. The molecular weight of this enzyme was determined using MALDI-TOF and size exclusion chromatography. PDH assays were employed to determine the kinetic parameters of the reaction catalyzed by the purified enzyme and to evaluate the effects of temperature, pH and ion concentration on PDH activity. Maximum PDH activity was obtained at 37 °C and pH 6.8 or below. Cations had little or no effect on PDH activity. Site-directed mutations were introduced into pdh. Amino acid replacements at conserved residues markedly reduced or eliminated enzyme activity. l-Tyrosine, a feedback inhibitor of PDH, showed strong inhibitory effects on PDH activity.