The rise of multidrug-resistant and totally drug-resistant tuberculosis and the association with an increasing number of HIV-positive patients developing tuberculosis emphasize the necessity to find new antitubercular targets and drugs. The antigen 85 (Ag85) complex from Mycobacterium tuberculosis plays important roles in the biosynthesis of major components of the mycobacterial cell envelope. For this reason, Ag85 has emerged as an attractive drug target. Recently, ebselen was identified as an effective inhibitor of the Ag85 complex through covalent modification of a cysteine residue proximal to the Ag85 active site and is therefore a covalent, allosteric inhibitor. To expand the understanding of this process, we have solved the x-ray crystal structures of Ag85C covalently modified with ebselen and other thiol-reactive compounds, p-chloromercuribenzoic acid and iodoacetamide, as well as the structure of a cysteine to glycine mutant. All four structures confirm that chemical modification or mutation at this particular cysteine residue leads to the disruption of the active site hydrogen-bonded network essential for Ag85 catalysis. We also describe x-ray crystal structures of Ag85C single mutants within the catalytic triad and show that a mutation of any one of these three residues promotes the same conformational change observed in the cysteine-modified forms. These results provide evidence for active site dynamics that may afford new strategies for the development of selective and potent Ag85 inhibitors.
Title: Structure of human epoxide hydrolase reveals mechanistic inferences on bifunctional catalysis in epoxide and phosphate ester hydrolysis Gomez GA, Morisseau C, Hammock BD, Christianson DW Ref: Biochemistry, 43:4716, 2004 : PubMed
The X-ray crystal structure of human soluble epoxide hydrolase (sEH) has been determined at 2.6 A resolution, revealing a domain-swapped quaternary structure identical to that observed for the murine enzyme [Argiriadi, M. A., Morisseau, C., Hammock, B. D., and Christianson, D. W. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10637-10642]. As with the murine enzyme, the epoxide hydrolytic mechanism of the human enzyme proceeds through an alkyl-enzyme intermediate with Asp-333 in the C-terminal domain. The structure of the human sEH complex with N-cyclohexyl-N'-(iodophenyl)urea (CIU) has been determined at 2.35 A resolution. Tyr-381 and Tyr-465 donate hydrogen bonds to the alkylurea carbonyl group of CIU, consistent with the proposed roles of these residues as proton donors in the first step of catalysis. The N-terminal domain of mammalian sEH contains a 15 A deep cleft, but its biological function is unclear. Recent experiments demonstrate that the N-terminal domain of human sEH catalyzes the metal-dependent hydrolysis of phosphate esters [Cronin, A., Mowbray, S., Durk, H., Homburg, S., Fleming, I., Fisslthaler, B., Oesch, F., and Arand, M. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 1552-1557; Newman, J. W., Morisseau, C., Harris, T. R., and Hammock, B. D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 1558-1563]. The binding of Mg(2+)-HPO4(2-) to the N-terminal domain of human sEH in its CIU complex reveals structural features relevant to those of the enzyme-substrate complex in the phosphatase reaction.
Title: Structural insights into ligand interactions at the acetylcholinesterase peripheral anionic site Bourne Y, Taylor P, Radic Z, Marchot P Ref: EMBO Journal, 22:1, 2003 : PubMed
The peripheral anionic site on acetylcholinesterase (AChE), located at the active center gorge entry, encompasses overlapping binding sites for allosteric activators and inhibitors; yet, the molecular mechanisms coupling this site to the active center at the gorge base to modulate catalysis remain unclear. The peripheral site has also been proposed to be involved in heterologous protein associations occurring during synaptogenesis or upon neurodegeneration. A novel crystal form of mouse AChE, combined with spectrophotometric analyses of the crystals, enabled us to solve unique structures of AChE with a free peripheral site, and as three complexes with peripheral site inhibitors: the phenylphenanthridinium ligands, decidium and propidium, and the pyrogallol ligand, gallamine, at 2.20-2.35 A resolution. Comparison with structures of AChE complexes with the peptide fasciculin or with organic bifunctional inhibitors unveils new structural determinants contributing to ligand interactions at the peripheral site, and permits a detailed topographic delineation of this site. Hence, these structures provide templates for designing compounds directed to the enzyme surface that modulate specific surface interactions controlling catalytic activity and non-catalytic heterologous protein associations.
