The phenylalanine-sensitive 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase (phe), a key enzyme involved in the biosynthesis of the aromatic amino acid phenylalanine, expressed by the Escherichia coli gene aroG, which catalyzes the condensation of d-erythrose 4-phosphate with phosphoenolpyruvate (PEP) to give DAH 7-P, was cloned into the expression vector pT7-7 for overexpression in E. coli. Purified enzyme from this expression system was used to demonstrate that DAH 7-P synthase (phe) also catalyses the aldol-type condensation of PEP with the 5-carbon analogues d-arabinose 5-phosphate, d-ribose 5-phosphate, and 2-deoxy-d-ribose 5-phosphate to yield 3-deoxy-d-manno-octulosonate 8-phosphate, 3-deoxy-d-altro-octulosonate 8-phosphate, and 3,5-dideoxy-d-gluco(manno)-octulosonate 8-phosphate, respectively, as determined by 1H NMR and other standard analytical methods. The kinetic parameters, K m and V max, for these reactions were determined. The 3- and 6-carbon phosphorylated monosaccharides, d,l-glyceraldehyde 3-phosphate and d-glucose 6-phosphate, as well as the nonphosphorylated 5-carbon analogues d-arabinose 5-phosphate, d-ribose 5-phosphate, and 2-deoxy-d-ribose 5-phosphate were not substrates.
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°C. The enzyme is extremely thermostable, with 50% of its initial activity retained after incubation for ϳ5 h at 80°C, 21 h at 70°C, and 86 h at 60°C. The enzyme appears to follow Michaelis-Menten kinetics with K m for phosphoenolpyruvate ؍ 9. The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) 1 synthase (EC 4.1.2.15) catalyzes the condensation of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) to form DAHP and inorganic phosphate. The formation of DAHP is the first committed step in the Shikimate pathway. This pathway is responsible for the biosynthesis of the intermediate compounds, chorismate and prephenate, which are precursors to the aromatic amino acids (Phe, Tyr, and Trp), catechols, and p-aminobenzoic acid (folic acid biosynthesis) as well as a number of other highly important microbial compounds (1).DAHP synthases exist in most microorganisms and plants. Based on phylogenetic analysis, this protein family has been separated into two classes, Class I and Class II, by Birck and Woodard (2). Alternatively, Jensen and co-workers (3, 4) classified DAHP synthases into two distinct homology families (AroA I and AroA II ). The AroA II family was defined as "plantlike" DAHP synthases that included the higher plant proteins and a cluster of microbial proteins (5). The AroA I family was further divided into subfamilies AroA I␣ and AroA I , which correspond to Class II and Class I, respectively (4). Escherichia coli expresses three DAHP synthase isoenzymes that are representative of Class II or the AroA I␣ family and require a divalent metal for activity (6). Each of the isoenzymes is specifically feedback-inhibited by only one of the three aromatic amino acids, Phe, Tyr, or Trp (7). The Bacillus subtilis DAHP synthase, which is representative of Class I or the AroA I family, is inhibited by the intermediates prephenate and chorismate in the Shikimate pathway and has been reported by Jensen and Nester (8, 9) to be insensitive to EDTA treatment and was thus proposed as a non-metalloenzyme (2).Based on the total lack of any information of a DAHP synthase from a thermophile, an investigation of a DAHP synthase from a thermophilic eubacterium was initiated in order to provide further insight into the biochemical reason for the bifurcation in the DAHP synthase phylogenetic tree. The extreme thermophile Thermotoga maritima DAHP synthase, which belongs to Class I (or the AroA I subfamily), was chosen for this study. Evolutionary studies have placed this bacterium in one of the deepest and most slowly evolving branches of the domain Bacteria (10, 11). Thus, studies on the DAHP synthase from this bacterium should provide a better understanding of the divergence of the two classes of DAHP synthase and t...
The active site residues of the proposed metal binding site of DAH 7-P synthase (phe) were probed by site-directed mutagenesis of C61 to glycine and serine, H64 to glycine, and with the double mutant C61H/H64C. While C61S and C61H/ H64C were inactive, both C61G and H64G were active. All mutants, regardless of enzymatic activity, bound one equivalent of Fe 2+ per monomeric unit. Even though C61 and H64 were shown not to be metal ligands for the DAH 7-P synthase (phe), they may provide some of the backbone interactions/secondary structural elements necessary to properly form the metal binding pocket.z 1998 Federation of European Biochemical Societies.
Escherichia coli 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8-P) synthase is able to utilize the five-carbon phosphorylated monosaccharide, 2-deoxyribose 5-phosphate (2dR5P), as an alternate substrate, but not D-ribose 5-phosphate (R5P) nor the four carbon analogue D-erythrose 4-phosphate (E4P). However, E. coli KDO8-P synthase in the presence of either R5P or E4P catalyzes the rapid consumption of approximately 1 mol of PEP per active site, after which consumption of PEP slows to a negligible but measurable rate. The mechanism of this abortive utilization of PEP was investigated using [2,3-(13)C(2)]-PEP and [3-F]-PEP, and the reaction products were determined by (13)C, (31)P, and (19)F NMR to be pyruvate, phosphate, and 2-phosphoglyceric acid (2-PGA). The formation of pyruvate and 2-PGA suggests that the reaction catalyzed by KDO8-P synthase may be initiated via a nucleophilic attack to PEP by a water molecule. In experiments in which the homologous enzyme, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7-P) synthase was incubated with D,L-glyceraldehyde 3-phosphate (G3P) and [2,3-(13)C(2)]-PEP, pyruvate and phosphate were the predominant species formed, suggesting that the reaction catalyzed by DAH7-P synthase starts with a nucleophilic attack by water onto PEP as observed in E. coli KDO8-P synthase.
The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase (Phe) is inactivated by diethyl pyrocarbonate (DEPC). The inactivation is first order with respect to enzyme and DEPC concentrations with a pseudo-second order rate constant of inactivation by DEPC of 4.9 ؎ 0.8 M ؊1 s ؊1 at pH 6.8 and 4°C. The dependence of inactivation on pH and the spectral features of enzyme modified at specific pH values imply that both histidine and cysteine residues are modified, which is confirmed by site-directed mutagenesis. Analysis of the chemical modification data indicates that one histidine is essential for activity. DAH 7-P synthase (Phe) is protected against DEPC inactivation by phosphoenolpyruvate, whereas D-erythrose 4-phosphate offers only minimal protection. The conserved residues H-172, H-207, H-268, and H-304 were individually mutated to glycine. The H304G and H207G mutants retain some level of activity, whereas the H268G and H172G mutants are virtually inactive. A comparison of the circular dichroism spectra of wild-type enzyme and the various mutants demonstrates that H-172 may play a structural role. Comparison of the UV spectra of the H268G and wildtype enzymes saturated with Cu 2؉ indicates that the metal-binding site of the H268G mutant resembles that of the wild-type enzyme. The residue H-268 may play a catalytic role based on the site-directed mutagenesis and spectroscopic studies. Cysteine 61 appears to influence the pK a of H-268 in the wild-type enzyme. The pK a of H-268 increases from 6.0 to 7.0 following mutation of C-61 to glycine.The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH 7-P) 1 synthase (Phe) (E.C. 4.1.2.15) catalyzes the formation of DAH 7-P (Scheme 1) from D-erythrose 4-phosphate (E 4-P) and phosphoenolpyruvate (PEP) (1, 2). The formation of DAH 7-P is the first committed step in the biosynthesis of phenylalanine, tyrosine, and tryptophan in microorganisms and plants as well as a plethora of other secondary biomolecules (1). The enzyme 3-deoxy-D-manno-octulosonate 8-phosphate (KDO 8-P) synthase (E.C. 4.1.2.16) catalyzes a similar reaction utilizing D-arabinose 5-phosphate and PEP to form KDO 8-P (3, 4). The formation of KDO 8-P is the first committed step in the biosynthesis of 3-deoxy-D-manno-octulosonate (KDO), which is an essential component of the lipopolysaccharide layer of Gram-negative bacteria (5, 6).Both enzymes, DAH 7-P synthase (Phe) and KDO 8-P synthase, catalyze aldol-like condensation reactions with the same facial selectivity with respect to the double bond of PEP and the aldehyde moiety of the monosaccharide substrate (7-10) as well as with cleavage of the C-O bond of PEP (9, 11-13) rather than the P-O bond. One significant difference between the two enzymes from Escherichia coli is that DAH 7-P synthase (Phe) requires a divalent metal and is regulated by a feedback inhibitor (14 -16), whereas KDO 8-P synthase neither requires a metal nor is sensitive to any known feedback inhibition (3).Sequence alignment studies of the DAH 7-P synthase (Phe) family ...
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