Thermotoga maritima 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase: the ancestral eubacterial DAHP synthase?
The gene encoding the 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase from the thermophilic microorganism Thermotoga maritima was cloned, and the enzyme was overexpressed in Escherichia coli. The purified DAHP synthase displays a homotetrameric structure and exhibits maximal activity at 90 degrees C. The enzyme is extremely thermostable, with 50% of its initial activity retained after incubation for approximately 5 h at 80 degrees C, 21 h at 70 degrees C, and 86 h at 60 degrees C. The enzyme appears to follow Michaelis-Menten kinetics with Km for phosphoenolpyruvate = 9.5-13 microm, Km for d-erythrose 4-phosphate = 57.3-350.1 microm, and kcat = 2.3-7.6 s-1 between 50 degrees C and 70 degrees C. Metal analysis indicates that DAHP synthase as isolated contains Zn2+, and the enzyme is inactivated by treatment with EDTA. The apo-enzyme is partially reactivated by a variety of divalent metals including Zn2+, Cd2+, Mn2+, Cu2+, Co2+, and Ni2+. These observations suggest that T. maritima DAHP synthase is a metalloenzyme. The activity of T. maritima DAHP synthase is inhibited by two of the three aromatic amino acids (l-Phe and l-Tyr) formed in the Shikimate pathway. This report is the first description of a thermophilic eubacterial DAHP synthase.
Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1065, USA.
Jing
J
Wu
David L
DL
Howe
Ronald W
RW
Woodard
D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011489:Protein Denaturation; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology*; D013997:Time Factors; D015032:Zinc / Q000737:chemistry
D007477:Ions; D004492:Edetic Acid; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D000446:Aldehyde-Lyases; D015032:Zinc