Insights into the Mechanism of 3-Deoxy-D-arabino-heptulosonate 7-Phosphate Synthase (Phe) from Escherichia coli Using a Transient Kinetic Analysis

Abstract

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 presteadystate 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.The first committed step in the biosynthesis of shikimic acid, which ultimately leads to the synthesis of aromatic amino acids, is catalyzed by 3-deoxy-arabino-heptulosonate 7-phosphate (DAHP) 1 synthase. The biosynthetic pathway of aromatic amino acids is absent in mammals, rendering the enzyme an attractive molecular target for antibiotic design. A putative inhibitor for the enzyme would inhibit bacterial growth while leaving the host organism unaffected.Over the years, DAHP synthase has been isolated from a wide range of organisms ranging from bacteria to eukaryotes (1-5). Escherichia coli harbors three DAHP synthase isozymes, each subject to feedback inhibition by one of the three aromatic amino acids (tyrosine, phenylalanine, and tryptophan, respectively). In the present study, we have focused on the phenylalanine-sensitive DAHP synthase encoded by E. coli aroG gene. The enzyme catalyzes the net aldol condensation of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) to form DAHP and inorganic phosphate (P i ) (Scheme 1).The degree of sequence homology between the E. coli DAHP synthase and the 3-deoxy-D-manno-2-octulosonic acid-8-phosphate (KDO8P synthase) and the conserved active site residues between the two enzymes allow us to extrapolate the mechanistic findings from one system to the other (6). ...

Intracellular Hydrogen Peroxide and Superoxide Poison 3-Deoxy-D-Arabinoheptulosonate 7-Phosphate Synthase, the First Committed Enzyme in the Aromatic Biosynthetic Pathway of Escherichia coli

“…Purified DAHP synthase was assayed as described previously (27). The purified enzyme was diluted 4-fold, thus reducing EDTA levels to 0.25 mM, and treated with 50 M tris(2-carboxyethyl)phosphine (TCEP) and 0.75 mM each metal in 0.1 M KP i (pH 6.4) under anoxic conditions for 10 min at ambient temperature.…”

Identification and Characterization of NeuB3 from Campylobacter jejuni as a Pseudaminic Acid Synthase

“…Formation of the tetrahedral intermediate was proposed to proceed in a stepwise fashion through an oxocarbenium ion intermediate. Two other well studied PEP condensing enzymes that utilize very similar mechanisms are 2-keto-3-deoxy-D-manno-octulosonate-8-phosphate synthase, which catalyzes the condensation of PEP and D-arabino-5-phosphate (17,18), and 2-keto-3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, which catalyzes the condensation of PEP and D-erythrose-4-phosphate (19,20). A key mechanistic experiment performed with all three of these synthases was the use of [2-18 O]PEP as a substrate during enzymatic incubations (see labeled atoms in Fig.…”

Mycobacterium tuberculosis Shikimate Pathway Enzymes as Targets for the Rational Design of Anti-Tuberculosis Drugs

“…The activity of EDTA-treated apo MtDAHPS could be restored in assays containing Co 2+ , Mn 2+ , Cd 2+ , Cu 2+ , Zn 2+ and Ca 2+ , with Co 2+ and Mn 2+ yielding the best results for restoring activity [28]. Furdui and coworkers [32] conducted experiments demonstrating that, in the case of E. coli DAHPS, the metal ion in the active site of the enzyme seems to play a structural role, orchestrating the arrangement of the active site residues in a position favorable for water activation. In this view, the geometry coordination of different metal ions may be a valuable factor for rational inhibitor design.…”