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Expression, purification, and characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Pyrococcus furiosus.
Protein Expr Purif. 2004 Mar; 34(1):17-27.PE

Abstract

The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P). DAH7PS from the hyperthermophile Pyrococcus furiosus has been expressed in Escherichia coli. The expressed protein was insoluble but was partially solubilized as a dimer by the inclusion of 200 mM KCl in the cell lysis buffer. An effective two step purification procedure has been developed. The first step resulted in a high degree of purification and involved lysis by sonication at approximately 40 degrees C followed by a heat treatment at 70 degrees C. A continuous assay measuring the loss of PEP at 232 nm at elevated temperatures was also developed. Temperature, pH, and divalent metal ions all had an effect on the extinction coefficient of PEP. Purified recombinant P. furiosus DAH7PS is a dimer with a subunit Mr of 29,226 (determined by ESMS), shows resistance to denaturation by SDS, has activity over a broad pH range, and has an activation energy of 88 kJmol-1. The kinetic parameters are Km (PEP) 120 microM, Km (E4P) 28 microM, and kcat 1.5s-1, at 60 degrees C and pH 6.8. DAH7PS is not inhibited by phenylalanine, tyrosine, or tryptophan. EDTA inactivates the enzyme and enzyme activity is restored by a wide range of divalent metal ions including (in order of decreasing effectiveness): Zn2+, Cd2+, Mn2+, Co2+, Ni2+, Ca2+, Hg2+, and Cu2+. This detailed characterization of the DAH7PS from P. furiosus raises the possibility that the subfamily Ibeta DAH7PS enzymes are metal ion dependent, contrary to previous predictions.

Authors+Show Affiliations

Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

14766297

Citation

Schofield, Linley R., et al. "Expression, Purification, and Characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate Synthase From Pyrococcus Furiosus." Protein Expression and Purification, vol. 34, no. 1, 2004, pp. 17-27.
Schofield LR, Patchett ML, Parker EJ. Expression, purification, and characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Pyrococcus furiosus. Protein Expr Purif. 2004;34(1):17-27.
Schofield, L. R., Patchett, M. L., & Parker, E. J. (2004). Expression, purification, and characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Pyrococcus furiosus. Protein Expression and Purification, 34(1), 17-27.
Schofield LR, Patchett ML, Parker EJ. Expression, Purification, and Characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate Synthase From Pyrococcus Furiosus. Protein Expr Purif. 2004;34(1):17-27. PubMed PMID: 14766297.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Expression, purification, and characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Pyrococcus furiosus. AU - Schofield,Linley R, AU - Patchett,Mark L, AU - Parker,Emily J, PY - 2003/08/28/received PY - 2003/11/06/revised PY - 2004/2/10/pubmed PY - 2004/10/27/medline PY - 2004/2/10/entrez SP - 17 EP - 27 JF - Protein expression and purification JO - Protein Expr Purif VL - 34 IS - 1 N2 - The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P). DAH7PS from the hyperthermophile Pyrococcus furiosus has been expressed in Escherichia coli. The expressed protein was insoluble but was partially solubilized as a dimer by the inclusion of 200 mM KCl in the cell lysis buffer. An effective two step purification procedure has been developed. The first step resulted in a high degree of purification and involved lysis by sonication at approximately 40 degrees C followed by a heat treatment at 70 degrees C. A continuous assay measuring the loss of PEP at 232 nm at elevated temperatures was also developed. Temperature, pH, and divalent metal ions all had an effect on the extinction coefficient of PEP. Purified recombinant P. furiosus DAH7PS is a dimer with a subunit Mr of 29,226 (determined by ESMS), shows resistance to denaturation by SDS, has activity over a broad pH range, and has an activation energy of 88 kJmol-1. The kinetic parameters are Km (PEP) 120 microM, Km (E4P) 28 microM, and kcat 1.5s-1, at 60 degrees C and pH 6.8. DAH7PS is not inhibited by phenylalanine, tyrosine, or tryptophan. EDTA inactivates the enzyme and enzyme activity is restored by a wide range of divalent metal ions including (in order of decreasing effectiveness): Zn2+, Cd2+, Mn2+, Co2+, Ni2+, Ca2+, Hg2+, and Cu2+. This detailed characterization of the DAH7PS from P. furiosus raises the possibility that the subfamily Ibeta DAH7PS enzymes are metal ion dependent, contrary to previous predictions. SN - 1046-5928 UR - https://www.unboundmedicine.com/medline/citation/14766297/Expression_purification_and_characterization_of_3_deoxy_D_arabino_heptulosonate_7_phosphate_synthase_from_Pyrococcus_furiosus_ DB - PRIME DP - Unbound Medicine ER -