Expression, purification, and characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Pyrococcus furiosus.

Published on Mar 1, 2004in Protein Expression and Purification1.291
· DOI :10.1016/j.pep.2003.11.008
Linley R. Schofield7
Estimated H-index: 7
(Massey University),
Mark L. Patchett19
Estimated H-index: 19
(Massey University),
Emily J. Parker22
Estimated H-index: 22
(Massey University)
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 °C followed by a heat treatment at 70 °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 M r 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 kJ mol −1 . The kinetic parameters are K m (PEP) 120 μM, K m (E4P) 28 μM, and k cat 1.5 s −1 , at 60 °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): Zn 2+ , Cd 2+ , Mn 2+ , Co 2+ , Ni 2+ , Ca 2+ , Hg 2+ , and Cu 2+ . This detailed characterization of the DAH7PS from P. furiosus raises the possibility that the subfamily Iβ DAH7PS enzymes are metal ion dependent, contrary to previous predictions.
  • References (44)
  • Citations (30)
📖 Papers frequently viewed together
47 Citations
53 Citations
47 Citations
78% of Scinapse members use related papers. After signing in, all features are FREE.
The β/α barrel is the common protein fold of numerous enzymes and was proposed recently to be the result of gene duplication and fusion of an ancient half-barrel. The initial enzyme of shikimate biosynthesis possesses the additional feature of feedback regulation. The crystal structure and kinetic studies on chimera and mutant proteins of yeast 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase from Saccharomyces cerevisiae inhibited by phenylalanine (Aro3p) and DAHP synthase S. cerevis...
65 CitationsSource
#1Chandra MohanH-Index: 1
98 Citations
#1Fabrizio GentileH-Index: 18
#2Pietro AmodeoH-Index: 25
Last. Roberto NucciH-Index: 22
view all 7 authors...
Abstract β-Glycosidases are fundamental, widely conserved enzymes. Those from hyperthermophiles exhibit unusual stabilities toward various perturbants. Previous work with homotetrameric β-glycosidase from hyperthermophilic Sulfolobus solfataricus (M r 226,760) has shown that addition of 0.05–0.1% SDS was associated with minimal secondary structure perturbations and increased activity. This work addresses the effects of SDS on β-glycosidase quaternary structure. In 0.1–1% SDS, the enzyme was dime...
44 CitationsSource
#1Roy A Jensen (CCNY: City College of New York)H-Index: 6
#2Gary Xie (LANL: Los Alamos National Laboratory)H-Index: 36
Last. Carol A. BonnerH-Index: 16
view all 4 authors...
-manno-octulosonate8-phosphate (KDOP) synthases.DAHP is the initial product that is specifically com-mitted to the biosynthesis of aromatic amino acids and avariety of other aromatic compounds via the action ofDAHP synthase. KDOP is best known as a key precursorof lipopolysaccharide in gram-negative bacteria, but itswider distribution in capsular polysaccharides, lipogly-can of Chlorella, and cell walls of higher plants impliesother functional roles at the cell surface as well [seeBrabetz et al....
45 CitationsSource
#1Emily J. ParkerH-Index: 22
Last. Chris AbellH-Index: 56
view all 4 authors...
3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS, EC catalyzes the condensation of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to give DAH7P via an ordered sequential mechanism. In the absence of PEP (the first substrate to bind), E4P binds covalently to the phenylalanine-sensitive DAH7PS of Escherichia coli, DAH7PS(Phe), deactivating the enzyme. Activity is restored on addition of excess PEP but not if deactivation was carried out in the presence of sodium c...
6 CitationsSource
#1Guillermo Gosset (UNAM: National Autonomous University of Mexico)H-Index: 39
#2Carol A. Bonner (UF: University of Florida)H-Index: 16
Last. Roy A. Jensen (UF: University of Florida)H-Index: 29
view all 3 authors...
Microorganisms and plants engage 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC as the initial catalyst for the commitment of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) to the biosynthesis of aromatic amino acids, vitamin K, folic acid, and ubiquinone (4). The pathway contributes heavily to the biochemical individuality of organisms, with an enormous variety of peripheral branch connections known. Various pigments, siderophores, signaling compounds, defe...
51 CitationsSource
#1Andrew McCarthy A (University of Auckland)H-Index: 28
#2Heather M. Baker (University of Auckland)H-Index: 37
Last. Edward N. Baker (University of Auckland)H-Index: 64
view all 7 authors...
Methylmalonyl-CoA epimerase (MMCE) is an enzyme that interconverts the R and S epimers of methylmalonyl-CoA in the pathway that links propionyl-CoA with succinyl-CoA. This is used for both biosynthetic and degradative processes, including the breakdown of odd-numbered fatty acids and some amino acids. The enzyme has been expressed in Escherichia coli both as the native enzyme and as its selenomethionine (SeMet) derivative. Crystals of both forms have been obtained by vapour diffusion using monom...
7 CitationsSource
#1Henry S. Duewel (UM: University of Michigan)H-Index: 7
#2Sergei Radaev (WSU: Wayne State University)H-Index: 5
Last. Domenico L. Gatti (WSU: Wayne State University)H-Index: 20
view all 5 authors...
Abstract 3-Deoxy-d-manno-octulosonate-8-phosphate synthase (KDO8PS) from the hyperthermophilic bacteriumAquifex aeolicus differs from its Escherichia coli counterpart in the requirement of a divalent metal for activity (Duewel, H. S., and Woodard, R. W. (2000)J. Biol. Chem. 275, 22824–22831). Here we report the crystal structure of the A. aeolicus enzyme, which was determined by molecular replacement using E. coli KDO8PS as a model. The structures of the metal-free and Cd2+ forms of the enzyme w...
55 CitationsSource
#1Matthew R. Birck (UM: University of Michigan)H-Index: 3
#2Ronald W. Woodard (UM: University of Michigan)H-Index: 27
The relationship between 3-deoxy-d-manno2-octulosonic acid 8-phosphate (KDO 8-P) synthase and 3-deoxy-d-arabino-2-heptulosonic acid 7-phosphate (DAH 7-P) synthase has not been adequately addressed in the literature. Based on recent reports of a metal requiring KDO 8-P synthase and the newly solved X-ray crystal structures of both Escherichia coli KDO 8-P synthase and DAH 7-P synthase, we begin to address the evolutionary kinship between these catalytically similar enzymes. Using a maximum likeli...
43 CitationsSource
#1Roy M. Daniel (University of Waikato)H-Index: 45
#2Michael J. DansonH-Index: 41
Publisher Summary There is now a wide variety of intra- and extracellular enzymes available from organisms growing above 75° and having sufficient stability to allow assay well above this temperature. For some of these enzymes, to assay below even 95° will involve measurement below the optimal growth temperature for the organism. This chapter covers practical aspects of enzyme assay procedures that are specific to high temperatures. The reason that by far the commonest routine assessment of enzy...
30 CitationsSource
Cited By30
#1Yasuyuki Yamamoto (Kyoto University)H-Index: 4
#2Tamotsu Kanai (Kyoto University)H-Index: 23
Last. Haruyuki Atomi (Kyoto University)H-Index: 45
view all 4 authors...
ABSTRACT TrpY from Methanothermobacter thermautotrophicus is a regulator that inhibits transcription of the Trp biosynthesis (trp) operon. Here, we show that the TrpY homolog in Thermococcus kodakarensis is not involved in such regulation. There are 87 genes on the T. kodakarensis genome predicted to encode transcriptional regulators (TRs). By screening for TRs that specifically bind to the promoter of the trp operon of T. kodakarensis, we identified TK0271. The gene resides in the aro operon, r...
#1Yu Bai (Victoria University of Wellington)H-Index: 1
#2Eric J. M. Lang (Cant.: University of Canterbury)H-Index: 3
Last. Emily J. Parker (Victoria University of Wellington)H-Index: 22
view all 4 authors...
: Because of their special organization, multifunctional enzymes play crucial roles in improving the performance of metabolic pathways. For example, the bacterium Prevotella nigrescens contains a distinctive bifunctional protein comprising a 3-deoxy-d-arabino heptulosonate-7-phosphate synthase (DAH7PS), catalyzing the first reaction of the biosynthetic pathway of aromatic amino acids, and a chorismate mutase (CM), functioning at a branch of this pathway leading to the synthesis of tyrosine and p...
#1Huaxian Zhao (Xida: Guangxi University)H-Index: 1
#2Hua Gao (Xida: Guangxi University)H-Index: 1
Last. Chengjian Jiang (Xida: Guangxi University)H-Index: 7
view all 11 authors...
3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) is a key rate-limiting enzyme in aromatic amino acid anabolism. A new Iβ-type DAHPS gene (aro1A) was identified in a metagenomic library from subtropical marine mangrove sediment. The gene encoded a polypeptide composed of 272 amino acids and had a maximum similarity of 52.4% to a known DAHPS at the amino acid level. Multiple sequence alignment, homologous modeling, and molecular docking showed that Aro1A had the typical (β/α)8 barrel-...
#1O.W. Sterritt (University of Auckland)H-Index: 1
#2Eric J. M. Lang (Cant.: University of Canterbury)H-Index: 3
Last. Emily J. Parker (Victoria University of Wellington)H-Index: 22
view all 7 authors...
In Pseudomonas aeruginosa ( Pae ), the shikimate pathway end product, chorismate, serves as the last common precursor for the biosynthesis of both primary aromatic metabolites, including phenylalanine, tyrosine and tryptophan, and secondary aromatic metabolites, including phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO). The enzyme 3-deoxy-d- arabino -heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, en route to chorismate. P. aeruginos...
1 CitationsSource
#1Anchal Sharma (IITR: Indian Institute of Technology Roorkee)H-Index: 3
#2Vijay Kumar (IITR: Indian Institute of Technology Roorkee)H-Index: 9
Last. Pravindra Kumar (IITR: Indian Institute of Technology Roorkee)H-Index: 16
view all 8 authors...
Abstract The first committed step of the shikimate pathway is catalyzed by a metalloenzyme 3-deoxy- d -arabino-heptulosonate-7-phosphate synthase (DAH7PS), which exhibits vulnerability to the oxidative stress. DAH7PS undergoes inactivation in multiple ways in the presence of redox metal, H 2 O 2 , and superoxide. The molecular mechanism and susceptibility of its inactivation might differ in different organisms and are presently unclear. In the present work, we have cloned, expressed and purified...
5 CitationsSource
#1Ali Reza Nazmi (Cant.: University of Canterbury)H-Index: 7
#2Eric J. M. Lang (Cant.: University of Canterbury)H-Index: 3
Last. Emily J. Parker (Cant.: University of Canterbury)H-Index: 22
view all 8 authors...
Abstract Multifunctional proteins play a variety of roles in metabolism. Here, we examine the catalytic function of the combined 3-deoxy-d-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM) from Geobacillus sp. DAH7PS operates at the start of the biosynthetic pathway for aromatic metabolites, whereas CM operates in a dedicated branch of the pathway for the biosynthesis of amino acids tyrosine and phenylalanine. In line with sequence predictions, the two catalytic func...
12 CitationsSource
#1Christy Rosaline Nirmal (SRM University)H-Index: 1
#2Rathankar Rao (SRM University)H-Index: 2
Last. Waheeta Hopper (SRM University)H-Index: 8
view all 3 authors...
Abstract Tuberculosis, caused by Mycobacterium tuberculosis , remains a serious global health threat, highlighting the urgent need for novel antituberculosis drugs. The shikimate pathway, responsible for aromatic amino acid biosynthesis, is required for the growth of Mycobacterium tuberculosis and is a potential drug target. 3-deoxy-D- arabino -heptulosonate 7-phosphate synthase (mtDAH7Ps) catalyzes the first step in shikimate pathway. E-pharmacophore models for inhibitors of mtDAH7Ps - tyrosine...
4 CitationsSource
#1William BainsH-Index: 14
#2Yao XiaoH-Index: 1
Last. Changyong YuH-Index: 1
view all 3 authors...
The components of life must survive in a cell long enough to perform their function in that cell. Because the rate of attack by water increases with temperature, we can, in principle, predict a maximum temperature above which an active terrestrial metabolism cannot function by analysis of the decomposition rates of the components of life, and comparison of those rates with the metabolites’ minimum metabolic half-lives. The present study is a first step in this direction, providing an analytical ...
9 CitationsSource
#1Ali Reza Nazmi (Cant.: University of Canterbury)H-Index: 7
#2Linley R. Schofield (Massey University)H-Index: 7
Last. Emily J. Parker (Cant.: University of Canterbury)H-Index: 22
view all 5 authors...
Abstract Many proteins adopt homomeric quaternary structures to support their biological function, including the first enzyme of the shikimate pathway that is ultimately responsible for the biosynthesis of the aromatic amino acids in plants and microorganisms. This enzyme, 3-deoxy- d - arabino -heptulosonate-7-phosphate synthase (DAH7PS), adopts a variety of different quaternary structures depending on the organism in which it is found. The DAH7PS from the hyperthermophilic archaebacterium Pyroc...
7 CitationsSource
In Arabidopsis wurden aufgrund von Sequenzhomologien drei Gene identifiziert, die putativ fur Isoformen des Eingangsenzyms des Shikimatwegs, die 3-Deoxy-D-arabino-heptulosonat 7-phosphat-Synthase (DAHPS) kodieren. In einer Reihe von Arbeiten konnte bereits gezeigt werden, dass diese Gene transkribiert und sehr unterschiedlich reguliert werden. In dieser Arbeit wurde nun erstmals durch heterologe Expression nachgewiesen, dass alle drei fur funktionelle DAHPS kodieren. Die Expressionsdaten weisen ...