Structure and characterization of the 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase from Aeropyrum pernix

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• 作者：Lily Zhou^b ; ^{mzhu48105@hotmail.com} ; Jing Wu^b ; ¹ ; ^{jingwu@jiangnan.edu.cn} ; Vijayalakshmi Janakiraman^b ; Igor A. Shumilin^a ; ² ; ^{igor@iwonka.med.virginia.edu} ; Ronald Bauerle^a ; ^{rhb7g@virginia.edu} ; Robert H. Kretsinger^a ; ^{rhk5i@virginia.edu} ; Ronald W. Woodard^b ; ^{rww@umich.edu}
• 关键词：BTP ; 1 ; 3-bis[tris(hydroxymethyl)methylamino]propane ; DAH7PS ; 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase ; DAH7PSAp ; the DAH7PS from Aeropyrum pernix ; DAH7PSPf ; the DAH7PS from Pyrococcus furiosus ; DAH7PSTm ; the DAH7PS from Thermatoga maritima ; DP
• 刊名：Bioorganic Chemistry
• 出版年：2012
• 出版时间：February 2012
• 年：2012
• 卷：40
• 期：Complete
• 页码：79-86
• 全文大小：1090 K

文摘

The first enzyme in the shikimic acid biosynthetic pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS), varies significantly in size and complexity in the bacteria and plants that express it. The DAH7PS from the archaebacterium Aeropyrum pernix (DAH7PS^Ap) is among the smallest and least complex of the DAH7PS enzymes, leading to the hypothesis that DAH7PS^Ap would not be subject to feedback regulation by shikimic acid pathway products. We overexpressed DAH7PS^Ap in Escherichia coli, purified it, and characterized its enzymatic activity. We then solved its X-ray crystal structure with a divalent manganese ion and phosphoenolpyruvate bound (PDB ID: ). DAH7PS^Ap is a homodimeric metalloenzyme in solution. Its enzymatic activity increases dramatically above 60 ¡ãC, with optimum activity at 95 ¡ãC. Its pH optimum at 60 ¡ãC is 5.7. DAH7PS^Ap follows Michaelis-Menten kinetics at 60 ¡ãC, with a K_M for erythrose 4-phosphate of 280 ¦ÌM, a K_M for phosphoenolpyruvate of 891 ¦ÌM, and a k_cat of 1.0 s^?. None of the downstream products of the shikimate biosynthetic pathway we tested inhibited the activity of DAH7PS^Ap. The structure of DAH7PS^Ap is similar to the structures of DAH7PS from Thermatoga maritima (PDB ID: ) and Pyrococcus furiosus (PDB ID: ), and is consistent with its designation as an unregulated DAH7PS.