Active site structure and mechanism of human glyoxalase I-an ab initio theoretical study.
ABSTRACT The structure of the active site of human glyoxalase I and the reaction mechanism of the enzyme-catalyzed conversion of the thiohemiacetal, formed from methylglyoxal and glutathione, to S-D-lactoylglutathione has been investigated by ab initio quantum chemical calculations. To realistically represent the environment of the reaction center, the effective fragment potential methodology has been employed, which allows systems of several hundred atoms to be described quantum mechanically. The methodology and the active site model have been validated by optimizing the structure of a known enzyme-inhibitor complex, which yielded structures in good agreement with the experiment. The same crystal structure has been used to obtain the quantum motif for the investigation of the glyoxalase I reaction. The results of our study confirm that the metal center of the active site zinc complex plays a direct catalytic role by binding the substrate and stabilizing the proposed enediolate reaction intermediate. In addition, our calculations yielded detailed information about the interactions of the substrate, the reaction intermediates, and the product with the active site of the enzyme and about the mechanism of the glyoxalase I reaction. The proton transfers of the reaction proceed via the two highly flexible residues Glu172 and Glu99. Information about the structural and energetic effect of the protein on the first-shell complex has been attained by comparison of the structures optimized in the local protein environment and in a vacuum. The environment of the zinc complex disturbs the Cs symmetry found for the complex in a vacuum, which suggests an explanation for the stereochemical behavior of glyoxalase I.
Article: Biosynthetic gene cluster of cetoniacytone A, an unusual aminocyclitol from the endosymbiotic Bacterium Actinomyces sp. Lu 9419.[show abstract] [hide abstract]
ABSTRACT: A gene cluster responsible for the biosynthesis of the antitumor agent cetoniacytone A was identified in Actinomyces sp. strain Lu 9419, an endosymbiotic bacterium isolated from the intestines of the rose chafer beetle (Cetonia aurata). The nucleotide sequence analysis of the 46 kb DNA region revealed the presence of 31 complete ORFs, including genes predicted to encode a 2-epi-5-epi-valiolone synthase (CetA), a glyoxalase/bleomycin resistance protein (CetB), an acyltransferase (CetD), an FAD-dependent dehydrogenase (CetF2), two oxidoreductases (CetF1 and CetG), two aminotransferases (CetH and CetM), and a pyranose oxidase (CetL). CetA has previously been demonstrated to catalyze the cyclization of sedoheptulose 7-phosphate to the cyclic intermediate, 2-epi-5-epi-valiolone. In this report, the glyoxalase/bleomycin resistance protein homolog CetB was identified as a 2-epi-5-epi-valiolone epimerase (EVE), a new member of the vicinal oxygen chelate (VOC) superfamily. The 24 kDa recombinant histidine-tagged CetB was found to form a homodimer; each monomer contains two betaalphabetabetabeta scaffolds that form a metal binding site with two histidine and two glutamic acid residues. A BLAST search using the newly isolated cet biosynthetic genes revealed an analogous suite of genes in the genome of Frankia alni ACN14a, suggesting that this plant symbiotic nitrogen-fixing bacterium is capable of producing a secondary metabolite related to the cetoniacytones.ChemBioChem 01/2009; 10(2):304-14. · 3.94 Impact Factor
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ABSTRACT: The structure and reactivity of the native, mutant, and metal substituted phosphotriesterase (PTE) is determined by ab initio quantum chemistry calculations. The x-ray structure for the Zn-Zn enzyme is leveraged into a catalytically competent active site in which a wide range of theoretical structures can be optimized for metal substituted and mutant active sites. The structural behavior of the active site is modeled using a new effective potential for representing the protein molecular environment (electrostatic, polarization, repulsive) interacting in the quantum Hamiltonian. The new methodology, effective fragment potentials (EFP), has been implemented in the GAMESS suite of electronic structure codes to make theoretical calculations on structure, spectroscopy, and reactivity tractable for systems involving many hundreds of atoms. Specific results on the structure of active site histidine to cysteine mutants, and a new proposal on the nucleophile for this hydrolase, will be presented.12/2001;
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ABSTRACT: Glyoxalase 2 is a beta-lactamase fold-containing enzyme that appears to be involved with cellular chemical detoxification. Although the cytoplasmic isozyme has been characterized from several organisms, essentially nothing is known about the mitochondrial proteins. As a first step in understanding the structure and function of mitochondrial glyoxalase 2 enzymes, a mitochondrial isozyme (GLX2-5) from Arabidopsis thaliana was cloned, overexpressed, purified, and characterized using metal analyses, EPR and (1)H NMR spectroscopies, and x-ray crystallography. The recombinant enzyme was shown to bind 1.04 +/- 0.15 eq of iron and 1.31 +/- 0.05 eq of Zn(II) and to exhibit k(cat) and K(m) values of 129 +/- 10 s(-1) and 391 +/- 48 microm, respectively, when using S-d-lactoylglutathione as the substrate. EPR spectra revealed that recombinant GLX2-5 contains multiple metal centers, including a predominant Fe(III)Z-n(II) center and an anti-ferromagnetically coupled Fe(III)Fe(II) center. Unlike cytosolic glyoxalase 2 from A. thaliana, GLX2-5 does not appear to specifically bind manganese. (1)H NMR spectra revealed the presence of at least eight paramagnetically shifted resonances that arise from protons in close proximity to a Fe(III)Fe(II) center. Five of these resonances arose from solvent-exchangeable protons, and four of these have been assigned to NH protons on metal-bound histidines. A 1.74-A resolution crystal structure of the enzyme revealed that although GLX2-5 shares a number of structural features with human GLX2, several important differences exist. These data demonstrate that mitochondrial glyoxalase 2 can accommodate a number of different metal centers and that the predominant metal center is Fe(III)Zn(II).Journal of Biological Chemistry 01/2006; 280(49):40668-75. · 4.77 Impact Factor