Cloning, sequencing, expression, purification and preliminary characterization of a type II dehydroquinase from Helicobacter pylori

Abstract

A heat-stable dehydroquinase was purified to near homogeneity from a plate-grown suspension of the Gram-negative stomach pathogen Helicobacter pylori, and shown from both its subunit and native molecular masses to be a member of the type II family of dehydroquinases. This was confirmed by N-terminal amino acid sequence data. The gene encoding this activity was isolated following initial identification, by random sequencing of the H. pylori genome, of a 96 bp fragment, the translated sequence of which showed strong identity to a C-terminal region of other type II enzymes. Southern blot analysis of a cosmid library identified several potential clones, one of which complemented an Escherichia coliaroD point mutant strain deficient in host dehydroquinase. The gene encoding the H. pylori type II dehydroquinase (designated aroQ) was sequenced. The translated sequence was identical to the N-terminal sequence obtained directly from the purified protein, and showed strong identity to other members of the type II family of dehydroquinases. The enzyme was readily expressed in E. coli from a plasmid construct from which several milligrams of protein could be isolated, and the molecular mass of the protein was confirmed by electrospray MS. The aroQ gene in H. pylori may function in the central biosynthetic shikimate pathway of this bacterium, thus opening the way for the construction of attenuated strains as potential vaccines as well as offering a new target for selective enzyme inhibition.

Systematic Identification of Selective Essential Genes in Helicobacter pylori by Genome Prioritization and Allelic Replacement Mutagenesis

“…The product of HP1038 (aroQ) has been purified and confirmed to be a type II dehydroquinase from the shikimate pathway (9). Disruption of shikimate pathway genes is well documented in other bacteria and has been shown to cause auxotrophy for aromatic amino acids and p-aminobenzoic acid, but we were unable to obtain HP1038 mutants on rich media even when these supplements were supplied exogenously.…”

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

“…Incorporation of [2][3][4][5][6][7][8][9][10][11][12][13] C]Histidine into Wild-type and Mutant Dehydroquinases-The plasmids pAL2, pAL5G, and pAL17 expressing wildtype, H143A, and H146A DHQases, respectively, were transformed into E. coli strain ATC1360 (⌬(gpt-proA)62, lacY1, tsx-29, supE44, galk2, Ϫ , aro D6, his G4(oc), xyl-5, mtl-1, arg E3, thi-1) obtained from the E. coli genetic stock center. A 50-ml seed flask of M9 minimal medium supplemented with arginine, proline (100 g/ml), [2-13 C]histidine (40 g/ml), thiamine (10 g/ml), and ampicillin (200 g/ml) was inoculated from a glycerol stock and grown for approximately 24 h. For the H143A protein preparations the medium was also supplemented with 200 g/ml shikimic acid.…”

“…Moreover, 4 of the 6 histidine residues in unliganded type I DHQase titrate in this pH range (2 of which, His-146 and peak b, were more or less complete titrations), but none of these displayed a pK a of 6.2 either. Each of the titrating peaks was fitted to a single ionizing curve described by the Henderson-Hasselbach equation; the pK a values that were determined were as follows: His-146, 6.84 (Ϯ0.05); peak a, 8.72 (Ϯ0.08); peak b, 7.61 (Ϯ0.04); peak c, 8.00 (Ϯ0.07). As for His-143, peak d did not titrate over the pH range studied.…”

“…It is therefore of great mechanistic and evolutionary interest to study the structure and mechanism of dehydroquinase enzymes to understand how this situation has arisen. Both types have been found in shikimate and quinate pathways, although there remain few examples of a type I DHQase in a quinate background, showing that the metabolic context of the reaction does not play a role in defining which class of DHQase is utilized (6).…”

Synthesis and Evaluation of Potent Ene–yne Inhibitors of Type II Dehydroquinases as Tuberculosis Drug Leads

“…[9,10] In contrast, the type II dehydroquinases are dodecamers that facilitate the anti-elimination of water via an E 1CB mechanism, whereby abstraction of the more acidic pro-S hydrogen (H S , Scheme 1) is facilitated by a conserved tyrosine residue, resulting in the formation of an enol intermediate 3 (Scheme 1). [7,11,12] Type II dehydroquinases are present in pathogenic bacteria such as Mycobacterium tuberculosis, [13] the etiological agent of tuberculosis (TB), and Helicobacter pylori, [14] a stomach pathogen that causes gastric ulcers and is linked to the development of stomach cancer. Due to the increased resistance of these and other microorganisms to current chemotherapies, antibacterial agents with novel mechanisms of action are required.…”