The Structure and Mechanism of the Type II Dehydroquinase from Streptomyces coelicolor

Abstract

The structure of the type II DHQase from Streptomyces coelicolor has been solved and refined to high resolution in complexes with a number of ligands, including dehydroshikimate and a rationally designed transition state analogue, 2,3-anhydro-quinic acid. These structures define the active site of the enzyme and the role of key amino acid residues and provide snap shots of the catalytic cycle. The resolution of the flexible lid domain (residues 21-31) shows that the invariant residues Arg23 and Tyr28 close over the active site cleft. The tyrosine acts as the base in the initial proton abstraction, and evidence is provided that the reaction proceeds via an enol intermediate. The active site of the structure of DHQase in complex with the transition state analog also includes molecules of tartrate and glycerol, which provide a basis for further inhibitor design.

Inhibition of Shikimate Kinase and Type II Dehydroquinase for Antibiotic Discovery: Structure-Based Design and Simulation Studies

“…In the absence of any crystal structure and based on kinetic isotope effects and pH profile studies carried out on Mt-DHQ2 and Aspergillus nidulans DHQ2 (An-DHQ2), Harris et al [41] suggested that the elimination proceeds by a stepwise E 1 CB mechanism via enolate intermediate 26 (Scheme 2). Subsequent resolution of the DHQ2 from Streptomyces coelicolor (Sc-DHQ2) with an inhibitor in the active site (PDB entry 1GU1, 1.8 Å) by Roszak et al [42] allowed a detailed description of the active site and provided a good knowledge of the specific functions of individual amino acid residues. The substrate is strongly bound to the active site by a series of hydrogen bonding interactions through the carboxylate group and the three hydroxyl groups.…”

“…WAT1 interacts through hydrogen bonding with a conserved asparagine (Asn10/Asn12 in Hp-DHQ2 and Mt-DHQ2, respectively), the carbonyl group of a conserved proline (Pro9/Pro11 in Hp-DHQ2 and Mt-DHQ2, respectively) and the main-chain amide of a glycine or an alanine (Ala79/Gly78 in Hp-DHQ2 and Mt-DHQ2, respectively). Initially, it was proposed that WAT1 would be involved in the enzymatic mechanism [42]. However, QM/MM studies suggest that WAT1 would be mainly involved in the stabilization of the substrate and enol intermediate 32 [44].…”

“…This substitution provides a more potent analog of 29, with K i values of 10 µM and 15 µM, respectively. The important observation that compounds with a double bond between C2-C3 bind in the manner predicted for transition state mimetics, as revealed by the crystal structure of Sc-DHQ2 with 29 bound in the active site (PDB entry 1GU1, 1.8 Å, [42]), inspired the structure-based design of new compounds with improved inhibition properties. Specifically, the 3-substituted cyclohexene derivatives 31-36, the tetrahydrobenzothiophene derivatives 37, the O-alkyl derivatives 38, and the 2,3-disubstituted compounds 39 were reported ( Fig.…”

“…The idea of adding substituents at the C3 position arose from the X-ray crystal structure of the Sc-DHQ2/29 binary complex (PDB entry 1GU1) reported by Roszak et al [42]. In the active site of this structure it was observed that, in addition to 29, a molecule each of glycerol and tartrate, originating from the enzyme storage buffer, were present at distances of 3.7 Å and 4.6 Å from the inhibitor 29, respectively.…”

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.…”