Structure and characterization of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Aeropyrum pernix.
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: 1VS1). DAH7PS(Ap) is a homodimeric metalloenzyme in solution. Its enzymatic activity increases dramatically above 60 °C, with optimum activity at 95 °C. Its pH optimum at 60 °C is 5.7. DAH7PS(Ap) follows Michaelis-Menten kinetics at 60 °C, with a K(M) for erythrose 4-phosphate of 280 μM, a K(M) for phosphoenolpyruvate of 891 μM, and a k(cat) of 1.0 s(-1). 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: 3PG8) and Pyrococcus furiosus (PDB ID: 1ZCO), and is consistent with its designation as an unregulated DAH7PS.
Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48105-1065, United States. Electronic address: mzhu48105@hotmail.com.
Lily
L
Zhou
Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48105-1065, United States. Electronic address: jingwu@jiangnan.edu.cn.
Jing
J
Wu
Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48105-1065, United States.
J
J
Vijayalakshmi
Department of Biology, University of Virginia, Charlottesville, VA 22903, United States. Electronic address: igor@iwonka.med.virginia.edu.
Igor A
IA
Shumilin
Department of Biology, University of Virginia, Charlottesville, VA 22903, United States. Electronic address: rhb7g@virginia.edu.
Ronald
R
Bauerle
Department of Biology, University of Virginia, Charlottesville, VA 22903, United States. Electronic address: rhk5i@virginia.edu.
Robert H
RH
Kretsinger
Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48105-1065, United States. Electronic address: rww@umich.edu.
Ronald W
RW
Woodard
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D041522:Aeropyrum / Q000201:enzymology*; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013403:Sugar Phosphates / Q000737:chemistry; D013696:Temperature
D011994:Recombinant Proteins; D013403:Sugar Phosphates; D008345:Manganese; C026959:erythrose 4-phosphate; D010728:Phosphoenolpyruvate; D010723:3-Deoxy-7-Phosphoheptulonate Synthase