Re-evaluating the Role of His-143 in the Mechanism of Type I Dehydroquinase from Escherichia coli Using Two-dimensional1H,13C NMR

Abstract

Type I dehydroquinase from the shikimate pathway of Escherichia coli dehydrates dehydroquinate to dehydroshikimate. pH/log V max profiles of the enzyme indicate the presence of a single ionizing group with a pK a of 6.2. Chemical modification experiments with diethyl pyrocarbonate have identified the conserved residue His-143 as essential for catalysis in this enzyme and the pK a for this modification is also 6.2, implying that this is the single ionizing residue in dehydroquinase that may be acting as a general base in the catalytic mechanism. Subsequent mutagenesis of this residue (Leech, A. P., James, R., Coggins, J. R., and Kleanthous, C. (1995) J. Biol. Chem. 270, 25827-25836) further suggested that His-143 may be involved in Schiff base formation/breakdown as well as being the proton abstracting general base. The importance of this residue was confirmed by recent x-ray crystallographic data showing His-143 to be at the center of a hydrogen-bonded triad, flanked by the essential Schiff base forming residue Lys-170 and Glu-86. In the present study, we have used mutagenesis and 1 H and 13 C NMR to assign the resonance of His-143 and probe its ionization state to define more precisely its role in the mechanism of type I dehydroquinase. Following isotopic enrichment of wild-type and H143A dehydroquinase enzymes with [2-13 C]histidine, the resonance for His-143 was assigned by comparing their 1 H, 13 C heteronuclear single quantum correlation NMR spectra. pH titrations revealed that whether in the liganded or unliganded state, His-143 does not ionize over the pH range 6 -9.5 and so cannot possess a pK a of 6.2. The NMR data are consistent with this residue remaining unprotonated at pH values optimal for the activity of this enzyme (pH > 7). The role of His-143 is re-evaluated in light of these and the recent structural data, and an alternative candidate for the pK a of 6.2 is discussed.The dehydration of 3-dehydroquinic acid to 3-dehydroshikimic acid is catalyzed by the enzyme 3-dehydroquinase (DHQase 1 ; EC 4.2.1.10) and occurs in two metabolic contexts, the biosynthetic shikimate pathway and the degradative quinate pathway. In a biosynthetic background, the enzyme introduces a double bond into the hexane ring of the substrate molecule, which is then converted through several enzymatic steps to produce the central metabolite, chorismic acid. From this precursor, numerous aromatic compounds are made including the aromatic amino acids, ubiquinone and vitamin E. The shikimate pathway is found in all prokaryotes and in lower eukaryotes such as fungi and plants but not in mammals and so is recognized as a source of potential targets for antimicrobial agents and herbicides (1). In a catabolic background, dehydroshikimate is further aromatized to produce protocatechuic acid that goes on to be metabolized to acetyl-CoA through the ␤-ketoadipate pathway (2). The quinate pathway, although best characterized in fungi, has also been found in prokaryotes such as Amylocaptosis methanolica and Acinetobacter calcoaceticus (3, ...

Structural and functional analysis of AsbF: Origin of the stealth 3,4-dihydroxybenzoic acid subunit for petrobactin biosynthesis

“…The nearby E142 could elevate basicity of H144 by forming a hydrogen bond to NE2 of H144, improving its proton abstraction ability. Histidine functions as a base for initial proton removal during many enzymatic reactions, including some type I 3-dehydroquinate dehydratases (23,24). Furthermore, although the activity of a H175A mutant is attenuated, the adjacent AsbF H144A mutant is completely inactive.…”

New insights into the mechanism of the Schiff base formation catalyzed by type I dehydroquinate dehydratase from S. enterica

“…On the basis of pH/log V max profiles [15] and the pK a value from diethyl polycarbonate treatment [16], a single ionizing group was measured with a pK a value of 6.2 and therefore postulated to be His143 residue. However, the pH titration indicated that His143 does not ionize over the pH range of 6-9.5 in the presence and absence of the binding substrate [17], and so cannot possess the pK a value of 6.2. Thus, the ionization observed with the pK a value of 6.2 should be associated with a neighboring residue of His143.…”

“…This enables the nucleophilic attack on the carbonyl carbon atom in the substrate by Lys170 residue. Furthermore, the K170A mutant does not affect the isoelectric point of the enzyme [17], meaning that Lys170 residue is likely uncharged. This supports that Lys170 is deprotonated with the low pK a value of 6.2 and is also in good agreement with the previously proposed mechanism [9].…”

“…The ionization states of His143 and Lys170 were set on the basis of the experimental studies [9,[15][16][17]. The ionization states of other ionizable residues were determined on the basis of the pK a value estimated by PROPKA program [18][19][20][21].…”

Progress in type II dehydroquinase inhibitors: From concept to practice

“…40,41 His143 is involved in Schiff base formation/hydrolysis, but its subsequent involvement in the dehydratation is uncertain. 42 Met23 and Met205 have also been identified as active site residues (near to the carboxylate of the substrate) although their role in substrate binding or catalysis has not yet been established. 43 …”