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1.
A plastid-localized isozytne of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, denoted DS-Mn, has been identified in a number of higher-plant species. Parallel characterizations were made of DS-Mn from Spinacia oleracea leaf tissue, Solanum tuberosum tubers, and Nicotiana silvestris suspension culture as sources of enzyme from plant materials which vary in phytogeny, developmental and tissue state, and physiological state. A highly conserved property of DS-Mn is a transition between inactive and active states, mediated by DTT as a hysteretic activator. A procedure for isolation of DS-Mn in the labile, inactive state is given. The process of activation appears to exhibit a higher pH optimum than the catalytic optimum. DTT-containing preparations are very stable. The enzyme characteristically exhibits stimulation by Mn++ in the range of 45–50%, relatively high affinity for erythrose-4-phosphate (E4P), dramatic substrate inhibition above about 0.5mol m?3 E4P, sigmoid substrate saturation curves for both E4P and phosphoenolpyruvate, and inhibition by L-arogenate (competitive against E4P and non-competitive against PEP). DS-Mn has a relatively high temperature optimum in the range of 45–50°C. Enzyme activity was lost when bound metal was stripped away by EDTA treatment. Reconstitution of the native-enzyme level of activity was obtained with Ca++, and additional stimulation was achieved with Mn++. DS-Mn control by L-arogenate in the chloroplast is proposed as one key circuit in an overall pattern of allosteric control for the entire pathway of aromatic amino acid biosynthesis. This pattern is called sequential feedback inhibition. The potential for modulation of this control system by environmental cues induced by light-dark transitions is discussed. 相似文献
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The activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS-Mn, DS-Co), phenylalanine ammonia-lyase (PAL),
and chalcone synthase (CHS) was monitored at various light intensities (dark, 8.88 μmol m−2 s−1, 88.8 μmol m−2 s−1) using a strawberry cell suspension culture. DS-Mn, PAL, and CHS were found to increase significantly (p>0.05) under light intensitie of 88.8 μmol m−2 s−1 compared to those of 8.88 μmol m−2 s−1 and dark. The activity of DS-Mn, PAL, and CHS were maximum at 88.8 μmol m−2 s−1. Anthocyanin content reached a maximum after 48–60 h of culturing at 88.8 μmol m−2 s−1. DS-Co showed greater activity than DS-Mn during cell culturing, but showed no correlation with anthocyanin production and
light intensity. The CHS gene expression was continuous at a light intensity of 88.8 μmol m−2 s−1.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
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The evolution of aromatic amino acid biosynthesis and its regulation is under study in a large assemblage of prokaryotes (Superfamily A) whose phylogenetic arrangement has been constructed on the criterion of oligonucleotide cataloging. One section of this Superfamily consists of a well defined (rRNA homology) cluster denoted as Group III pseudomonads. Pseudomonas acidovorans ATCC 11299a, a Group III member, was chosen for indepth studies of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase, the initial regulatory enzyme of aromatic biosynthesis. This strain is of particular interest for evolutionary studies of aromatic metabolism because it possesses phenylalanine hydroxylase, an enzyme whose physiological role and distribution among prokaryotes is largely unknown. Although P. acidovorans ATCC 11299a has been of uncertain identity, we now establish it unambiguously as a species of acidovorans by virtue of its 87% DNA homology with P. acidovorans ATCC 15668 (type strain). This result conformed with enzyme patterning studies which placed ATCC 11299a into pseudomonad Group IIIa, a subgroup containing the acidovorans species. Crude extracts of Group III pseudomonads had previously been shown to share, as a common group characteristic, sensitivity of DAHP synthase to feedback inhibition by either l-tyrosine or l-phenylalanine. Detailed studies with partially purified preparations from strain ATCC 11299a revealed the presence of two distinct regulatory isozymes, DAHP synthase-phe and DAHP synthase-tyr. DAHP synthase-tyr is tightly controlled by l-tyrosine with 50% inhibition of activity being achieved at 4.0 M effector. DAHP synthase-phe is inhibited 50% by 40 M
l-phenylalanine and exhibits dramatic changes in levels of activity, as well as chromatographic elution patterns, in response to dithiothreitol. This two-isozyme pattern of DAHP synthase has not been described previously, although it may prove to be widespread.Abbreviations DAHP
3-deoxy-d-arabino-heptulosonate 7-phosphate
- E4P
d-erythrose-4-phosphate
- PEP
phosphoenolpyruvate
- DTT
dithiothreitol
- BSA
fraction V bovine serum albumin 相似文献
6.
The effect of inactivation of the glucose phosphotransferase transport system (PTS) on 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) productivity and yield from glucose in Escherichia coli is reported. Strains used in this study were the PTS(+) PB103 and its PTS(-) glucose(+) derivative NF9. Their aroB(-) derivatives PB103B and NF9B were constructed to allow accurate measurement of total carbon flow into the aromatic pathway. The measured specific rates of DAHP synthesis were 0.55 and 0.94 mmol/g-dcw. h and the DAHP molar yields from glucose were 0.43 and 0.71 mol/mol for the PTS(+) aroB(-)and the PTS(-) glucose(+) aroB(-)strains, respectively. For the latter strain, this value represents 83% of the maximum theoretical yield for DAHP synthesis from glucose. 相似文献
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The cloning and nucleotide sequence of a Corynebacterium glutamicum 3-deoxy-d- arabinoheptulosonate-7-phosphate synthase gene 总被引:2,自引:0,他引:2
Abstract The aro gene of Corynebacterium glutamucum CCRC 18310 encoding 3-deoxy- d -arabinoheptulosonate-7-phosphate (DAHP) synthase was isolated by complementation of a DAHP synthase-deficient mutant of Escherichia coli AB3257. The specific activity of DAHP synthase was increased four-fold in a C. glutamicum strain harboring the cloned aro gene. The complete nucleotide sequence of the aro gene and 5' and 3' flanking regions has been determined. The sequence contained an open reading frame of 368 codons, from which a protein with a molecular mass of 39 340 Da could be predicted. The deduced amino acid sequence shows high identity with the aro gene products of E. coli and Salmonella typhimurium . 相似文献
8.
Ron Lou Doong John E. Gander Robert J. Ganson Roy A. Jensen 《Physiologia plantarum》1992,84(3):351-360
The cytosolic isoenzyme of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS-Co: EC 4.1.2.15) in Spinacia oleracea, Solanum tubersosum and many other higher plants was found to use a diversity of substrates. Diose (glycolaldehyde), triose (D-glyceraldehyde, L-glyceraldehyde and DL-glyceraldehyde 3-phosphate), tetrose (D-erythrose, L-erythrose, D-erythrose 4-phosphate, D-threose and L-threose), and pentose (D-ribose 5-phosphate and D-arabinose 5-phosphate) were utilized in combination with phosphoenolpyruvate (PEP) to make the corresponding 2-keto-3-deoxy sugar acids. Glyoxylate was also utilized by DS-Co. Glycoladehyde exhibited the highest reaction velocity when substrates were tested at 3 mM concentrations. Pentoses were poor substrates except when phsophorylated, an effect which is probably due to an increased fraction of the molecules being in the open-chain form. Little stereoselective discrimination exists since comparable velocities were demonstrated with the D and L isomers of glyceraldehyde, erythrose or threose. The enzyme is not a reversible aldolase since pyruvate failed to substitute for PEP. The use of D-erythrose 4-phsophate or glycolaldehyde resulted in Km values of 1.95 mM and 8.60 mM, respectively. However, glycolaldehyde exhibited the largest VmaxKm ratio, suggesting a greater catalytic efficiency for this substrate. Glycolaldehyde is an ideal substrate for inexpensive assays of DS-Co that are absolutely selective in the presence of two other plant enzymes which also utilize erythrose 4-phosphate and PEP. The spinach DS-Co enzymes required divalent metals for activity. The presence of 20 mM Mg2+, 1 mM Co2+ and 1 mM Mn2+ yielded relative activities of 100, 70 and 15, respectively. The pH optimum was 9.5 and temperature optimum for activity was 49°C. The molecular masses of DS-Co from spinach, tobacco and pea were all in the range of 400 kDa. The possible roles of DS-Co in biosynthesis of α-ketoglutarate and aromatic amino acids, in biosynthesis of components of cell wall and phytotoxin, and in acting as a sink for 2-and 3-carbon sugars are discussed. 相似文献
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Tyrosine feedback-inhibits the 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase isoenzyme AroF of Escherichia coli. Here we show that an Asn-8 to Lys-8 substitution in AroF leads to a tyrosine-insensitive DAHP synthase. This mutant enzyme exhibited similar activities (v=30-40 U mg(-1)) and substrate affinities (K(m)(erythrose-4-phosphate)=0.5 mM, positive cooperativity with respect to phospho(enol)pyruvate) as the wild-type AroF, but showed decreased thermostability. An engineered AroF enzyme lacking the seven N-terminal residues also was tyrosine-resistant. These results strongly suggest that the N-terminus of AroF is involved in the molecular interactions occurring in the feedback-inhibition mechanism. 相似文献
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The phenylalanine-regulated isozyme of 3-deoxy-D-arabino-heptulosonate-7-phosphate- synthase (DAHPS) from Escherichia coli, its binary complexes with either substrate, phosphoenolpyruvate (PEP), or feedback inhibitor, Phe, and its ternary complexes with either PEP or Phe plus metal cofactor (either Mn2+, Cd2+, or Pb2+) were crystallized from polyethylglycol (PEG) solutions. All crystals of the DAHPS without Phe belong to space group C2, with cell parameters a = 213.5 Å, b = 54.3 Å, c = 149.0 Å, β = 116.6°. All crystals of the enzyme with Phe also belong to space group C2, but with cell parameters a = 297.1 Å, b = 91.4 Å, c = 256.5 Å, and β = 148.2°. 相似文献
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3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthases are metal-dependent enzymes that catalyse the first committed step in the biosynthesis of aromatic amino acids in microorganisms and plants, the condensation of 2-phophoenolpyruvate (PEP) and d-erythrose 4-phosphate (E4P) to DAHP. The DAHP synthases are possible targets for fungicides and represent a model system for feedback regulation in metabolic pathways. To gain further insight into the role of the metal ion and the catalytic mechanism in general, the crystal structures of several complexes between the tyrosine-regulated form of DAHP synthase from Saccharomyces cerevisiae and different metal ions and ligands have been determined. The crystal structures provide evidence that the simultaneous presence of a metal ion and PEP result in an ordering of the protein into a conformation that is prepared for binding the second substrate E4P. The site and binding mode of E4P was derived from the 1.5A resolution crystal structure of DAHP synthase in complex with PEP, Co2+, and the E4P analogue glyceraldehyde 3-phosphate. Our data suggest that the oxygen atom of the reactive carbonyl group of E4P replaces a water molecule coordinated to the metal ion, strongly favouring a reaction mechanism where the initial step is a nucleophilic attack of the double bond of PEP on the metal-activated carbonyl group of E4P. Mutagenesis experiments substituting specific amino acids coordinating PEP, the divalent metal ion or the second substrate E4P, result in stable but inactive Aro4p-derivatives and show the importance of these residues for the catalytic mechanism. 相似文献
13.
Light SH Halavaty AS Minasov G Shuvalova L Anderson WF 《Protein science : a publication of the Protein Society》2012,21(6):887-895
3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) catalyzes the first step in the biosynthesis of a number of aromatic metabolites. Likely because this reaction is situated at a pivotal biosynthetic gateway, several DAHPS classes distinguished by distinct mechanisms of allosteric regulation have independently evolved. One class of DAHPSs contains a regulatory domain with sequence homology to chorismate mutase-an enzyme further downstream of DAHPS that catalyzes the first committed step in tyrosine/phenylalanine biosynthesis-and is inhibited by chorismate mutase substrate (chorismate) and product (prephenate). Described in this work, structures of the Listeria monocytogenes chorismate/prephenate regulated DAHPS in complex with Mn(2+) and Mn(2+) + phosphoenolpyruvate reveal an unusual quaternary architecture: DAHPS domains assemble as a tetramer, from either side of which chorismate mutase-like (CML) regulatory domains asymmetrically emerge to form a pair of dimers. This domain organization suggests that chorismate/prephenate binding promotes a stable interaction between the discrete regulatory and catalytic domains and supports a mechanism of allosteric inhibition similar to tyrosine/phenylalanine control of a related DAHPS class. We argue that the structural similarity of chorismate mutase enzyme and CML regulatory domain provides a unique opportunity for the design of a multitarget antibacterial. 相似文献
14.
Christian Triantaphylides Louis Nespoulous Christian Chervia 《Physiologia plantarum》1993,87(3):389-395
The effects of radiation-mediated free radical production on polyamine metabolism were investigated in grape cells ( Vitis vinifera L. cv. Gamay) using a cell suspension culture. Putrescine (Put) synthesis was triggered in irradiated cells (0. 5 kGy) only when ammonium was present in the culture medium. Under these conditions. Put accumulated to a continuously high level. As also described for other kinds of stress, the level of spermidine was slightly enhanced and that of spermine unchanged. The role of ammonium was assessed by studying non-irradiated cell cultures. In the presence of ammonium, a transient increase of both arginine decarboxylase (ADC) activity and of Put synthesis was observed during the lag phase of growth. This Put enhancement was inhibited by difluoromethyl arginine and not by difluoromethyl ornithine, showing that increased Put synthesis occurs via the ADC pathway. When ammonium was withheld from the culture medium. ADC activity was still triggered though transient Put accumulation was completely suppressed. These results emphasize the importance of ammonium availability in cultured cells as a limiting factor for Put production. Polyamine synthesis, therefore, cannot be stimulated by gamma irradiation in the absence of an ammonium supply. These results support the hypothesis that Put synthesis is a detoxification process of the ammonium produced as a result of nitrogen recycling within stressed plant cells. 相似文献
15.
Structure of the amplified 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate-resistant carrot cells 总被引:3,自引:0,他引:3
The structure of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) DNA of carrot suspension-cultured cell lines selected for glyphosate resistance was analyzed to determine the mechanism of gene amplification in this plant system. Southern hybridization of the amplified DNA digested with several restriction enzymes probed with a petunia EPSPS cDNA clone showed that there were differences in fragment sizes in the amplified DNA from one highly resistant cell line in comparison with the parental line. Cloning of the EPSPS gene and 5 flanking sequences was carried out and two different DNA structures were revealed. A 13 kb clone contained only one copy of the EPSPS gene while a 16 kb clone contained an inverted duplication of the gene. Southern blot analysis with a carrot DNA probe showed that only the uninverted repeated DNA structure was present in all of the cell lines during the selection process and the inverted repeat (IR) was present only in highly amplified DNA. The two structures were present in about equal amounts in the highly amplified line, TC 35G, where the EPSPS gene was amplified about 25-fold. The presence of the inverted repeat (IR) was further verified by resistance to S1 nuclease hydrolysis after denaturation and rapid renaturation, showing foldback DNA with the IR length being 9.5 kb. The junction was also sequenced. Mapping of the clones showed that the size of the amplified carrot EPSPS gene itself is about 3.5 kb. This is the first report of an IR in amplified DNA of a target enzyme gene in selected plant cells. 相似文献
16.
Furdui C Zhou L Woodard RW Anderson KS 《The Journal of biological chemistry》2004,279(44):45618-45625
Escherichia coli phenylalanine-sensitive 3-deoxy-arabino-heptulosonate 7-phosphate synthase (DAHP synthase) catalyzes the net aldol condensation of phosphoenolpyruvate and erythrose 4-phosphate to form 3-deoxy-D-arabino-heptulosonate 7-phosphate and inorganic phosphate. For the first time, the presteady-state kinetic analysis of the Phe-sensitive DAHP synthase from E. coli is reported. The steady-state and presteady-state kinetic parameters of the DAHP synthase reconstituted with Mn(II), Cu(II), and Zn(II) were compared. These studies showed the following: 1) product release is rate-limiting for all of the three metal ions studied under physiologically relevant conditions; 2) concentration of the active sites of the metal-containing DAHP synthase is increasing from Mn- (30%) to Zn- (52%) and to Cu-DAHP synthase (88%); 3) rate constant for product formation is higher in Mn- (130-200 s(-1)) than Cu- (55 s(-1)) and Zn-DAHP synthase (6.8 s(-1)); and 4) steady-state rate (rate constant for product release) is higher for the Mn- (70 s(-1)) than for Cu- (5.6 s(-1)) and Zn-DAHP synthase (1.8 s(-1)). In addition, an examination of the reaction kinetics at lower pH reveals that for Cu-DAHP synthase, product release is no longer rate-limiting, whereas the Mn- and Zn-DAHP synthase show a slower rate of product formation, suggesting that the intermediate formation becomes rate-limiting in product formation. Also, a deuterium-isotope effect on the burst rate constant of product formation for Mn-DAHP synthase was observed at pH 6.0. This supports the hypothesis that the role of metal ion in E. coli DAHP synthase is to position the amino acids with the appropriate geometry required to coordinate and activate the water molecule. 相似文献
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Evolution of the regulatory isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase present in the Escherichia coli genealogy. 总被引:1,自引:1,他引:0
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The evolutionary history of isozymes for 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase has been constructed in a phylogenetic cluster of procaryotes (superfamily B) that includes Escherichia coli. Members of superfamily B that have been positioned on a phylogenetic tree by oligonucleotide cataloging possess one or more of four distinct isozymes of DAHP synthase. DAHP synthase-0 is insensitive to feedback inhibition, while DAHP synthase-Tyr, DAHP synthase-Trp, and DAHP synthase-Phe are sensitive to feedback inhibition by L-tyrosine, L-tryptophan, and L-phenylalanine, respectively. The evolutionary history of this isozyme family can be deduced within superfamily B by using a cladistic methodology of maximum parsimony (R. A. Jensen, Mol. Biol. Evol. 2:92-108, 1985). DAHP synthase-0 was found in Acinetobacter species and in Oceanospirillum minutulum, organisms that also possess DAHP synthase-Tyr. These two isozymes were apparently present in a common ancestor that predated the evolutionary divergence of contemporary superfamily B sublineages. DAHP synthase-0 is postulated to have been the evolutionary forerunner of DAHP synthase-Trp. The newly evolved DAHP synthase-Trp is postulated to have possessed sensitivity to feedback inhibition by chorismate as well as by L-tryptophan, chorismate sensitivity having been retained in rRNA group I pseudomonads (minor sensitivity), group V pseudomonads (very sensitive), and Lysobacter enzymogenes (ultrasensitive). Organisms constituting the enteric lineage of the phylogenetic tree (including a cluster of four Oceanospirillum species) have all lost the chorismate sensitivity of DAHP synthase-Trp. The absence of DAHP synthase-Phe in the Oceanospirillum cluster of organisms supports the previous conclusion that DAHP synthase-Phe evolved recently within superfamily B, being present only Escherichia coli and its close relatives. 相似文献
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3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS), the first enzyme of the aromatic biosynthetic pathway in microorganisms and plants, catalyzes the aldol-like condensation of phosphoenolpyruvate and D-erythrose-4-phosphate with the formation of 3-deoxy-D-arabino-heptulosonate-7-phosphate. In Escherichia coli, there are three isoforms of DAHPS, each specifically feedback-regulated by one of the three aromatic amino acid end products. The crystal structure of the phenylalanine-regulated DAHPS from E.coli in complex with its inhibitor, L-phenylalanine, phosphoenolpyruvate, and metal cofactor, Mn(2+), has been determined to 2.8A resolution. Phe binds in a cavity formed by residues of two adjacent subunits and is located about 20A from the closest active site. A model for the mechanism of allosteric inhibition has been derived from conformational differences between the Phe-bound and previously determined Phe-free structures. Two interrelated paths of conformational changes transmit the inhibitory signal from the Phe-binding site to the active site of DAHPS. The first path involves transmission within a single subunit due to the movement of adjacent segments of the protein. The second involves alterations in the contacts between subunits. The combination of these two paths changes the conformation of one of the active site loops significantly and shifts the other slightly. This alters the interaction of DAHPS with both of its substrates. Upon binding of Phe, the enzyme loses the ability to bind D-erythrose-4-phosphate and binds phosphoenolpyruvate in a flipped orientation. 相似文献