Periplasmic ß-glucosidase BglX from E. coli demonstrates greater activity towards galactose-containing substrates.

BACKGROUND: The diverse nature of carbohydrate structures and linkages requires a variety of enzymes responsible for sugar degradation. The E. coli periplasmic protein encoded by the bglX gene has been assigned to glycoside hydrolase family 3 and is predicted to function as a ß-glucosidase. OBJECTIVES: We investigated the catalytic properties of the E. coli protein BglX and identified two functionally important amino acid residues. METHODS: The bglX gene was cloned into a pET20b(+) vector, and three mutants, D111N, D287G, and E293Q, were generated using site-directed mutagenesis. Kinetic studies were performed on the wild-type and mutant enzymes. RESULTS: Substrate specificity tests indicated that the BglX enzyme hydrolyzes ß-glycosidic bonds in nitrophenyl-ß-glycosides and demonstrates greater activity towards galactose-containing substrates compared to glucose derivatives. Monomeric glucose and galactose inhibit enzyme activity to a different degree in a substrate-dependent manner. In addition, BglX can hydrolyze lactose but not cellobiose, maltose, or laminarin. Subsequently, E. coli cells overexpressing active BglX have a growth advantage on minimal media supplemented with lactose as a carbon source. Mutation of D287 or D111 residues negatively affected the activity of BglX indicating their involvement in catalysis. Overexpression of BglX by E. coli cells did not increase biofilm formation. CONCLUSIONS: The low activity towards glucose-containing substrates and significantly elevated activity towards galactosides suggests that ß-glucosidase activity may not be the primary function of the BglX enzyme.

Comparison of the functions of glutathionylspermidine synthetase/amidase from E. coli and its predicted homologues YgiC and YjfC.

Protein function prediction is very important in establishing the roles of various proteins in bacteria; however, some proteins in the E. coli genome have their function assigned based on low percent sequence homology that does not provide reliable assignments. We have made an attempt to verify the prediction that E. coli genes ygiC and yjfC encode proteins with the same function as glutathionylspermidine synthetase/amidase (GspSA). GspSA is a bifunctional enzyme that catalyzes the ATP-dependent formation and hydrolysis of glutathionylspermidine (G-Sp), a conjugate of glutathione (GSH) and spermidine. YgiC and YjfC proteins show 51% identity between themselves and 28% identity to the synthetase domain of the GspSA enzyme. YgiC and YjfC proteins were expressed and purified, and the properties of GspSA, YgiC, and YjfC were compared. In contrast to GspSA, proteins YgiC and YjfC did not bind to G-Sp immobilized on the affinity matrix. We demonstrated that all three proteins (GspSA, YgiC and YjfC) catalyze the hydrolysis of ATP; however, YgiC and YjfC cannot synthesize G-Sp, GSH, or GSH intermediates. gsp, ygiC, and yjfC genes were eliminated from the E. coli genome to test the ability of mutant strains to synthesize G-Sp conjugate. E. coli cells deficient in GspSA do not produce G-Sp while synthesis of the conjugate is not affected in ΔygiC and ΔyjfC mutants. All together our results indicate that YgiC and YjfC are not glutathionylspermidine synthetases as predicted from the amino acid sequence analysis.

Structure and function of YghU, a nu-class glutathione transferase related to YfcG from Escherichia coli.

The crystal structure (1.50 Å resolution) and biochemical properties of the GSH transferase homologue, YghU, from Escherichia coli reveal that the protein is unusual in that it binds two molecules of GSH in each active site. The crystallographic observation is consistent with biphasic equilibrium binding data that indicate one tight (K(d1) = 0.07 ± 0.03 mM) and one weak (K(d2) = 1.3 ± 0.2 mM) binding site for GSH. YghU exhibits little or no GSH transferase activity with most typical electrophilic substrates but does possess a modest catalytic activity toward several organic hydroperoxides. Most notably, the enzyme also exhibits disulfide-bond reductase activity toward 2-hydroxyethyl disulfide [k(cat) = 74 ± 6 s(-1), and k(cat)/K(M)(GSH) = (6.6 ± 1.3) × 10(4) M(-1) s(-1)] that is comparable to that previously determined for YfcG. A superposition of the structures of the YghU·2GSH and YfcG·GSSG complexes reveals a remarkable structural similarity of the active sites and the 2GSH and GSSG molecules in each. We conclude that the two structures represent reduced and oxidized forms of GSH-dependent disulfide-bond oxidoreductases that are distantly related to glutaredoxin 2. The structures and properties of YghU and YfcG indicate that they are members of the same, but previously unidentified, subfamily of GSH transferase homologues, which we suggest be called the nu-class GSH transferases.

Analysis of the structure and function of YfcG from Escherichia coli reveals an efficient and unique disulfide bond reductase.

YfcG is one of eight glutathione (GSH) transferase homologues encoded in the Escherichia coli genome. The protein exhibits low or no GSH transferase activity toward a panel of electrophilic substrates. In contrast, it has a very robust disulfide-bond reductase activity toward 2-hydroxyethyldisulfide on par with mammalian and bacterial glutaredoxins. The structure of YfcG at 2.3 A-resolution from crystals grown in the presence of GSH reveals a molecule of glutathione disulfide in the active site. The crystallographic results and the lack of functional cysteine residues in the active site of YfcG suggests that the reductase activity is unique in that no sulfhydryl groups in the YfcG protein are covalently involved in the redox chemistry.

Conformational dynamics in the F/G segment of CYP51 from Mycobacterium tuberculosis monitored by FRET.

A cysteine was introduced into the FG-loop (P187C) of CYP51 from Mycobacterium tuberculosis (MT) for selective labeling with BODIPY and fluorescence energy transfer (FRET) analysis. Förster radius for the BODIPY-heme pair was calculated assuming that the distance between the heme and Cys187 in solution corresponds to that in the crystal structure of ligand free MTCYP51. Interaction of MTCYP51 with azole inhibitors ketoconazole and fluconazole or the substrate analog estriol did not influence the fluorescence, but titration with the substrate lanosterol quenched BODIPY emission, the effect being proportional to the portion of substrate bound MTCYP51. The detected changes correspond to approximately 10A decrease in the calculated distance between BODIPY-Cys187 and the heme. The results confirm (1) functional importance of conformational motions in the MTCYP51 F/G segment and (2) applicability of FRET to monitor them in solution.

Silencing of the Tropomyosin-1 gene by DNA methylation alters tumor suppressor function of TGF-beta.

Loss of actin stress fibers has been associated with cell transformation and metastasis. TGF-beta induction of stress fibers in epithelial cells requires high molecular weight tropomyosins encoded by TPM1 and TPM2 genes. Here, we investigated the mechanism underlying the failure of TGF-beta to induce stress fibers and inhibit cell migration in metastatic cells. RT-PCR analysis in carcinoma cell lines revealed a significant reduction in TPM1 transcripts in metastatic MDA-MB-231, MDA-MB-435 and SW620 cell lines. Treatment of these cells with demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) increased mRNA levels of TPM1 with no effect on TPM2. Importantly, 5-aza-dC treatment of MDA-MB-231 cells restored TGF-beta induction of TPM1 and formation of stress fibers. Forced expression of TPM1 by using Tet-Off system increased stress fibers in MDA-MB-231 cells and reduced cell migration. A potential CpG island spanning the TPM1 proximal promoter, exon 1, and the beginning of intron 1 was identified. Bisulfite sequencing showed significant cytosine methylation in metastatic cell lines that correlated with a reduced expression of TPM1. Together these results suggest that epigenetic suppression of TPM1 may alter TGF-beta tumor suppressor function and contribute to metastatic properties of tumor cells.

Smad3-ATF3 signaling mediates TGF-beta suppression of genes encoding Phase II detoxifying proteins.

This study provides evidence that in mammary epithelial cells the pluripotent cytokine TGF-beta1 repressed expression of multiple genes involved in Phase II detoxification. GCLC, the gene that encodes the catalytic subunit of the enzyme glutamate cysteine ligase, the rate-limiting enzyme in the biosynthesis of glutathione, was used as a molecular surrogate for investigating the mechanisms by which TGF-beta suppressed Phase II gene expression. TGF-beta was found to suppress luciferase reporter activity mediated by the human GCLC proximal promoter, as well as reporter activity mediated by the GCLC antioxidant response element, ARE4. TGF-beta downregulated expression of endogenous GCLC mRNA and GCLC protein. TGF-beta suppression of the Phase II genes correlated with a decrease in cellular glutathione and an increase in cellular reactive oxygen species. Ectopic expression of constitutively active Smad3E was sufficient to inhibit both reporters in the absence of TGF-beta, whereas dominant negative Smad3A blocked TGF-beta suppression. Smad3E suppressed Nrf2-mediated activation of the GCLC reporter. We demonstrate that TGF-beta increased ATF3 protein levels, as did transient overexpression of Smad3E. Ectopic expression of ATF3 was sufficient to suppress the GCLC reporter activity, as well as endogenous GCLC expression. These results demonstrate that Smad3-ATF3 signaling mediates TGF-beta repression of ARE-dependent Phase II gene expression and potentially provide critical insight into mechanisms underlying TGF-beta1 function in carcinogenesis, tissue repair, and fibrosis.

Catalytic mechanism of dichloromethane dehalogenase from Methylophilus sp. strain DM11.

The glutathione (GSH)-dependent dichloromethane dehalogenase from Methylophilus sp. strain DM11 catalyzes the dechlorination of CH(2)Cl(2) to formaldehyde via a highly reactive, genotoxic intermediate, S-(chloromethyl)glutathione (GS-CH(2)Cl). The catalytic mechanism of the enzyme toward a series of dihalomethane and monohaloethane substrates suggests that the initial addition of GSH to the alkylhalides is fast and that the rate-limiting step in turnover is the release of either the peptide product or formaldehyde. With the exception of CH(2)ClF, which forms a relatively stable GS-CH(2)F intermediate, the turnover numbers for a series of dihalomethanes fall in a very narrow range (1-3 s(-1)). The pre-steady-state kinetics of the DM11-catalyzed addition of GSH to CH(3)CH(2)Br exhibits a burst of S-(ethyl)-glutathione (k(b) = 96 +/- 56 s(-1)) followed by a steady state with k(cat) = 0.13 +/- 0.01 s(-1). The turnover numbers for CH(3)CH(2)Cl, CH(3)CH(2)Br, and CH(3)CH(2)I are identical, indicating a common rate-limiting step. The turnover numbers of the enzyme with CH(3)CH(2)Br and CH(3)CH(2)I are dependent on viscosity and are very close to the measured off-rate of GSEt. The turnover number with CH(2)I(2) is also dependent on viscosity, suggesting that a diffusive step is rate-limiting with dihaloalkanes as well. The rate constants for solvolysis of CH(3)SCH(2)Cl, a model for GS-CH(2)Cl, range between 1 s(-1) (1:1 dioxane/water) and 64 s(-1) (1:10 dioxane/water). Solvolysis of the S-(halomethyl)glutathione intermediates may also occur in the active site of the enzyme preventing the release of the genotoxic species. Together, the results indicate that dissociation of the GS-CH(2)X or GS-CH(2)OH intermediates from the enzyme may be a relatively rare event.

Local protein dynamics and catalysis: detection of segmental motion associated with rate-limiting product release by a glutathione transferase.

Glutathione transferase rGSTM1-1 catalyzes the addition of glutathione (GSH) to 1-chloro-2,4-dinitrobenzene, a reaction in which the chemical step is 60-fold faster than the physical step of product release. The hydroxyl group of Y115, located in the active site access channel, controls the egress of product from the active site. The Y115F mutant enzyme has a k(cat) (72 s(-)(1)) that is 3.6-fold larger than that of the native enzyme (20 s(-)(1)). Crystallographic observations and evidence from amide proton exchange kinetics are consistent with localized increases in the degree of segmental motion of the Y115F mutant that are coupled to the enhanced rate of product release. The loss of hydrogen bonding interactions involving the hydroxyl group of Y115 is reflected in subtle alterations in the backbone position, an increase in B-factors for structural elements that comprise the channel to the active site, and, most dramatically, a loss of well-defined electron density near the site of mutation. The kinetics of amide proton exchange are also enhanced by a factor between 3 and 12 in these regions, providing direct, quantitative evidence for changes in local protein dynamics affecting product release. The enhanced product release rate is proposed to derive from a small shift in the equilibrium population of protein conformers that permit egress of the product from the active site.