Bacterial Glutathione S-transferase Research Articles

CcpN: a moonlighting protein regulating catabolite repression of gluconeogenic genes in Bacillus subtilis also affects cell length and interacts with DivIVA.

CcpN is a transcriptional repressor in Bacillus subtilis that binds to the promoter region of gapB and pckA, downregulating their expression in the presence of glucose. CcpN also represses sr1, which encodes a small noncoding regulatory RNA that suppresses the arginine biosynthesis gene cluster. CcpN has homologues in other Gram-positive bacteria, including Enterococcus faecalis. We report the interaction of CcpN with DivIVA of B. subtilis as determined using bacterial two-hybrid and glutathione S-transferase pull-down assays. Insertional inactivation of CcpN leads to cell elongation and formation of straight chains of cells. These findings suggest that CcpN is a moonlighting protein involved in both gluconeogenesis and cell elongation.

Structural and biochemical characterization of a glutathione transferase from the citrus canker pathogen Xanthomonas.

The genus Xanthomonas comprises several cosmopolitan plant-pathogenic bacteria that affect more than 400 plant species, most of which are of economic interest. Citrus canker is a bacterial disease that affects citrus species, reducing fruit yield and quality, and is caused by the bacterium Xanthomonas citri subsp. citri (Xac). The Xac3819 gene, which has previously been reported to be important for citrus canker infection, encodes an uncharacterized glutathione S-transferase (GST) of 207 amino-acid residues in length (XacGST). Bacterial GSTs are implicated in a variety of metabolic processes such as protection against chemical and oxidative stresses. XacGST shares high sequence identity (45%) with the GstB dehalogenase from Escherichia coli O6:H1 strain CFT073 (EcGstB). Here, XacGST is reported to be able to conjugate glutathione (GSH) with bromoacetate with a Km of 6.67 ± 0.77 mM, a kcat of 42.69 ± 0.32 s-1 and akcat/Km of 6.40 ± 0.72 mM-1 s-1 under a saturated GSH concentration (3.6 mM). These values are comparable to those previously reported for EcGstB. In addition, crystal structures of XacGST were determined in the apo form (PDB entry 6nxv) and in a GSH-bound complex (PDB entry 6nv6). XacGST has a canonical GST-like fold with a conserved serine residue (Ser12) at the GSH-binding site near the N-terminus, indicating XacGST to be a serine-type GST that probably belongs to the theta-class GSTs. GSH binding stabilizes a loop of about 20 residues containing a helix that is disordered in the apo XacGST structure.

Delineation of the functional and structural properties of the glutathione transferase family from the plant pathogen Erwinia carotovora

Erwinia carotovora, a widespread plant pathogen that causes soft rot disease in many plants, is considered a major threat in agriculture. Bacterial glutathione transferases (GSTs) play important roles in a variety of metabolic pathways and processes, such as the biodegradation of xenobiotics, protection against abiotic stress, and resistance against antimicrobial drugs. The GST family of canonical soluble enzymes from Erwinia carotovora subsp. atroseptica strain SCRI1043 (EcaGSTs) was investigated. Genome analysis showed the presence of six putative canonical cytoplasmic EcaGSTs, which were revealed by phylogenetic analysis to belong to the well-characterized GST classes beta, nu, phi, and zeta. The analysis also revealed the presence of two isoenzymes that were phylogenetically close to the omega class of GSTs, but formed a distinct class. The EcaGSTs were cloned and expressed in Escherichia coli, and their catalytic activity toward different electrophilic substrates was elucidated. The EcaGSTs catalyzed different types of reactions, although all enzymes were particularly active in reactions involving electrophile substitution. Gene and protein expression profiling conducted under normal culture conditions as well as in the presence of the herbicide alachlor and the xenobiotic 1-chloro-2,4-dinitrobenzene (CDNB) showed that the isoenzyme EcaGST1, belonging to the omega-like class, was specifically induced at both the protein and mRNA levels. EcaGST1 presumably participates in counteracting the xenobiotic toxicity and/or abiotic stress conditions, and may therefore represent a novel molecular target in the development of new chemical treatments to control soft rot diseases.

Characterization of a Highly pH Stable Chi-Class Glutathione S-Transferase from Synechocystis PCC 6803.

Glutathione S-transferases (GSTs) are multifunctional enzymes present in virtually all organisms. Besides having an essential role in cellular detoxification, they also perform various other functions, including responses in stress conditions and signaling. GSTs are highly studied in plants and animals; however, the knowledge regarding GSTs in cyanobacteria seems rudimentary. In this study, we report the characterization of a highly pH stable GST from the model cyanobacterium- Synechocystis PCC 6803. The gene sll0067 was expressed in Escherichia coli (E. coli), and the protein was purified to homogeneity. The expressed protein exists as a homo-dimer, which is composed of about 20 kDa subunit. The results of the steady-state enzyme kinetics displayed protein’s glutathione conjugation activity towards its class specific substrate- isothiocyanate, having the maximal activity with phenethyl isothiocyanate. Contrary to the poor catalytic activity and low specificity towards standard GST substrates such as 1-chloro-2,4-dinitrobenzene by bacterial GSTs, PmGST B1-1 from Proteus mirabilis, and E. coli GST, sll0067 has broad substrate degradation capability like most of the mammalian GST. Moreover, we have shown that cyanobacterial GST sll0067 is catalytically efficient compared to the best mammalian enzymes. The structural stability of GST was studied as a function of pH. The fluorescence and CD spectroscopy in combination with size exclusion chromatography showed a highly stable nature of the protein over a broad pH range from 2.0 to 11.0. To the best of our knowledge, this is the first GST with such a wide range of pH related structural stability. Furthermore, the presence of conserved Proline-53, structural motifs such as N-capping box and hydrophobic staple further aid in the stability and proper folding of cyanobacterial GST- sll0067.

An alternate pathway of arsenate resistance in E. coli mediated by the glutathione S-transferase GstB.

Microbial arsenate resistance is known to be conferred by specialized oxidoreductase enzymes termed arsenate reductases. We carried out a genetic selection on media supplemented with sodium arsenate for multicopy genes that can confer growth to E. coli mutant cells lacking the gene for arsenate reductase (E. coli ΔarsC). We found that overexpression of glutathione S-transferase B (GstB) complemented the ΔarsC allele and conferred growth on media containing up to 5 mM sodium arsenate. Interestingly, unlike wild type E. coli arsenate reductase, arsenate resistance via GstB was not dependent on reducing equivalents provided by glutaredoxins or a catalytic cysteine residue. Instead, two arginine residues, which presumably coordinate the arsenate substrate within the electrophilic binding site of GstB, were found to be critical for transferase activity. We provide biochemical evidence that GstB acts to directly reduce arsenate to arsenite with reduced glutathione (GSH) as the electron donor. Our results reveal a pathway for the detoxification of arsenate in bacteria that hinges on a previously undescribed function of a bacterial glutathione S-transferase.

Functional classification and biochemical characterization of a novel rho class glutathione S-transferase in Synechocystis PCC 6803

We report a novel class of glutathione S-transferase (GST) from the model cyanobacterium Synechocystis PCC 6803 (sll1545) which catalyzes the detoxification of the water pollutant dichloroacetate and also shows strong glutathione-dependent peroxidase activity representing the classical activities of zeta and theta/alpha class respectively. Interestingly, sll1545 has very low sequence and structural similarity with these classes. This is the first report of dichloroacetate degradation activity by any bacterial GST. Based on these results we classify sll1545 to a novel GST class, rho. The present data also indicate potential biotechnological and industrial applications of cyanobacterial GST in dichloroacetate-polluted areas.

Stereochemical Features of Glutathione-dependent Enzymes in the Sphingobium sp. Strain SYK-6 β-Aryl Etherase Pathway

Glutathione-dependent enzymes play important protective, repair, or metabolic roles in cells. In particular, enzymes in the glutathione S-transferase (GST) superfamily function in stress responses, defense systems, or xenobiotic detoxification. Here, we identify novel features of bacterial GSTs that cleave β-aryl ether bonds typically found in plant lignin. Our data reveal several original features of the reaction cycle of these GSTs, including stereospecific substrate recognition and stereoselective formation of β-S-thioether linkages. Products of recombinant GSTs (LigE, LigP, and LigF) are β-S-glutathionyl-α-keto-thioethers that are degraded by a β-S-thioetherase (LigG). All three Lig GSTs produced the ketone product (β-S-glutathionyl-α-veratrylethanone) from an achiral side chain-truncated model substrate (β-guaiacyl-α-veratrylethanone). However, when β-etherase assays were conducted with a racemic model substrate, β-guaiacyl-α-veratrylglycerone, LigE- or LigP-catalyzed reactions yielded only one of two potential product (β-S-glutathionyl-α-veratrylglycerone) epimers, whereas the other diastereomer (differing in configuration at the β-position (i.e. its β-epimer)) was produced only in the LigF-catalyzed reaction. Thus, β-etherase catalysis causes stereochemical inversion of the chiral center, converting a β(R)-substrate to a β(S)-product (LigE and LigP), and a β(S)-substrate to a β(R)-product (LigF). Further, LigG catalyzed glutathione-dependent β-S-thioether cleavage with β-S-glutathionyl-α-veratrylethanone and with β(R)-configured β-S-glutathionyl-α-veratrylglycerone but exhibited no or significantly reduced β-S-thioether-cleaving activity with the β(S)-epimer, demonstrating that LigG is a stereospecific β-thioetherase. We therefore propose that multiple Lig enzymes are needed in this β-aryl etherase pathway in order to cleave the racemic β-ether linkages that are present in the backbone of the lignin polymer.

Identification and clarification of the role of key active site residues in bacterial glutathione S-transferase zeta/maleylpyruvate isomerase

The maleylpyruvate isomerase NagL from Ralstonia sp. strain U2, which has been structurally characterized previously, catalyzes the isomerization of maleylpyruvate to fumarylpyruvate. It belongs to the class zeta glutathione S-transferases (GSTZs), part of the cytosolic GST family (cGSTs). In this study, site-directed mutagenesis was conducted to probe the functions of 13 putative active site residues. Steady-state kinetic information for mutants in the reduced glutathione (GSH) binding site, suggested that (a) Gln64 and Asp102 interact directly with the glutamyl moiety of glutathione, (b) Gln49 and Gln64 are involved in a potential electron-sharing network that influences the ionization of the GSH thiol. The information also suggests that (c) His38, Asn108 and Arg109 interact with the GSH glycine moiety, (d) His104 has a role in the ionization of the GSH sulfur and the stabilization of the maleyl terminal carboxyl group in the reaction intermediate and (e) Arg110 influences the electron distribution in the active site and therefore the ionization of the GSH thiolate. Kinetic data for mutants altered in the substrate-binding site imply that (a) Arg8 and Arg176 are critical for maleylpyruvate orientation and enolization, and (b) Arg109 (exclusive to NagL) participates in k cat regulation. Surprisingly, the T11A mutant had a decreased GSH K m value, whereas little impact on maleylpyruvate kinetics was observed, suggesting that this residue plays an important role in GSH binding. An evolutionary trend in this residue appears to have developed not only in prokaryotic and eukaryotic GSTZs, but also among the wider class of cGSTs.

Bioinformatic analysis and in vitro site-directed mutagenesis of conserved amino acids in BphK LB400, a specific bacterial glutathione transferase

Glutathione transferases [GSTs: EC 2.5.1.18)] are ubiquitous multifunctional prokaryotic and eukaryotic enzymes involved in the cellular detoxification and excretion of a large variety of compounds. However, our understanding of the role of bacterial GSTs in metabolism is still in its infancy. The association of bacterial GST DNA with other genes involved in degradation of toxic pollutants, including polychlorinated biphenyls (PCBs), indirectly suggests a role for bacterial GSTs in biodegradation. Previously, in this laboratory, a specific bacterial GST, BphK LB400 isolated from Burkholderia xenovorans LB400, was shown to be capable of dehalogenating chlorinated organic substrates rendering them less toxic. However, little is known about the specific amino acids in BphK LB400 involved in catalysis in vitro. In this study, bioinformatic analysis of BphK LB400 and other bacterial GSTs, including PCB degraders, identified a number of amino acids that were identical in all bacterial GST sequences analysed. Two amino acids, Cys10 and His106, were selected for in vitro site-directed mutagenesis studies. In vitro GST activity assay results suggest that these two amino acids play a role in determining the catalytic activity of BphK LB400. Studies of bacterial cell extracts expressing BphK LB400 (wildtype and mutant) identified a specific mutant, Cys10Phe, with increased GST activity towards 1-chloro-2,4-dinitrobenzene (the model substrate for GSTs). BphK LB400 (mutant) with increased activity towards toxic chlorinated organic compounds could have potential for bioremediation of contaminated soil in the environment.

Crystallization and preliminary X-ray analysis of glutathione transferases from cyanobacteria.

Glutathione S-transferases (GSTs) are a group of multifunctional enzymes that are found in animals, plants and microorganisms. Their primary function is to remove toxins derived from exogenous sources or the products of metabolism from the cell. Mammalian GSTs have been extensively studied, in contrast to bacterial GSTs which have received relatively scant attention. A new class of GSTs called Chi has recently been identified in cyanobacteria. Chi GSTs exhibit a high glutathionylation activity towards isothiocyanates, compounds that are normally found in plants. Here, the crystallization of two GSTs are presented: TeGST produced by Thermosynechococcus elongates BP-1 and SeGST from Synechococcus elongates PCC 6301. Both enzymes formed crystals that diffracted to high resolution and appeared to be suitable for further X-ray diffraction studies. The structures of these GSTs may shed further light on the evolution of GST catalytic activity and in particular why these enzymes possess catalytic activity towards plant antimicrobial compounds.

Glutathione transferases in bacteria

Bacterial glutathione transferases (GSTs) are part of a superfamily of enzymes that play a key role in cellular detoxification. GSTs are widely distributed in prokaryotes and are grouped into several classes. Bacterial GSTs are implicated in a variety of distinct processes such as the biodegradation of xenobiotics, protection against chemical and oxidative stresses and antimicrobial drug resistance. In addition to their role in detoxification, bacterial GSTs are also involved in a variety of distinct metabolic processes such as the biotransformation of dichloromethane, the degradation of lignin and atrazine, and the reductive dechlorination of pentachlorophenol. This review article summarizes the current status of knowledge regarding the functional and structural properties of bacterial GSTs.

Structure of Bacterial Glutathione-S-Transferase Maleyl Pyruvate Isomerase and Implications for Mechanism of Isomerisation

Maleyl pyruvate isomerase (MPI) is a bacterial glutathione S-transferase (GST) from the pathway for degradation of naphthalene via gentisate that enables the bacterium Ralstonia to use polyaromatic hydrocarbons as a sole carbon source. Genome sequencing projects have revealed the presence of large numbers of GSTs in bacterial genomes, often located within gene clusters encoding the degradation of different aromatic compounds. This structure is therefore an example of this under-represented class of enzymes. Unlike many glutathione transferases, the reaction catalysed by MPI is an isomerisation of an aromatic ring breakdown product, and glutathione is a true cofactor rather than a substrate in the reaction. We have solved the structure of the enzyme in complex with dicarboxyethyl glutathione, an analogue of a proposed reaction intermediate, at a resolution of 1.3 Å. The structure provides direct evidence that the glutathione thiolate attacks the substrate in the C2 position, with the terminal carboxylate buried at the base of the active site cleft. Our structures suggest that the C1–C2 bond remains fixed so when rotation occurs around the C2–C3 bond the atoms from C4 onwards actually move. We identified a conserved arginine that is likely to stabilize the enolate form of the substrate during the isomerisation. Arginines at either side of the active site cleft can interact with the end of the substrate/product and preferentially stabilise the product. MPI has significant sequence similarity to maleylacetoacetate isomerase (MAAI), which performs an analogous reaction in the catabolism of phenylalanine and tyrosine. The proposed mechanism therefore has relevance to the MAAIs. Significantly, whilst the overall sequence identity is 40% only one of the five residues from the Zeta motif in the active site is conserved. We re-examined the roles of the residues in the active site of both enzymes and the Zeta motif itself.

A Novel Nuclear Interactor of ARF and MDM2 (NIAM) That Maintains Chromosomal Stability

The ARF tumor suppressor signals through p53 and other poorly defined anti-proliferative pathways to block carcinogenesis. In a search for new regulators of ARF signaling, we discovered a novel nuclear protein that we named NIAM (nuclear interactor of ARF and MDM2) for its ability to bind both ARF and the p53 antagonist MDM2. NIAM protein is normally expressed at low to undetectable levels in cells because of, at least in part, MDM2-mediated ubiquitination and proteasomal degradation. When reintroduced into cells, NIAM activated p53, caused a G1 phase cell cycle arrest, and collaborated with ARF in an additive fashion to suppress proliferation. Notably, NIAM retains growth inhibitory activity in cells lacking ARF and/or p53, and knockdown experiments revealed that it is not essential for ARF-mediated growth inhibition. Thus, NIAM and ARF act in separate anti-proliferative pathways that intersect mechanistically and suppress growth more effectively when jointly activated. Intriguingly, silencing of NIAM accelerated chromosomal instability, and microarray analyses showed reduced NIAM mRNA expression in numerous primary human tumors. This study identifies a novel protein with tumor suppressor-like behaviors and functional links to ARF-MDM2-p53 signaling.

Structures of Ternary Complexes of BphK, a Bacterial Glutathione S-Transferase That Reductively Dechlorinates Polychlorinated Biphenyl Metabolites

Prokaryotic glutathione S-transferases are as diverse as their eukaryotic counterparts but are much less well characterized. BphK from Burkholderia xenovorans LB400 consumes two GSH molecules to reductively dehalogenate chlorinated 2-hydroxy-6-oxo-6-phenyl-2,4-dienoates (HOPDAs), inhibitory polychlorinated biphenyl metabolites. Crystallographic structures of two ternary complexes of BphK were solved to a resolution of 2.1A. In the BphK-GSH-HOPDA complex, GSH and HOPDA molecules occupy the G- and H-subsites, respectively. The thiol nucleophile of the GSH molecule is positioned for SN2 attack at carbon 3 of the bound HOPDA. The respective sulfur atoms of conserved Cys-10 and the bound GSH are within 3.0A, consistent with product release and the formation of a mixed disulfide intermediate. In the BphK-(GSH)2 complex, a GSH molecule occupies each of the two subsites. The three sulfur atoms of the two GSH molecules and Cys-10 are aligned suitably for a disulfide exchange reaction that would regenerate the resting enzyme and yield disulfide-linked GSH molecules. A second conserved residue, His-106, is adjacent to the thiols of Cys-10 and the GSH bound to the G-subsite and thus may stabilize a transition state in the disulfide exchange reaction. Overall, the structures support and elaborate a proposed dehalogenation mechanism for BphK and provide insight into the plasticity of the H-subsite.

Evolutionarily conserved structural motifs in bacterial GST (glutathione S-transferase) are involved in protein folding and stability

The bacterium Proteus mirabilis expresses a cytosolic class beta glutathione S-transferase (PmGST B1-1) that is part of a family of multifunctional detoxication enzymes. Like other cytosolic GSTs, PmGST B1-1 possesses two local structural motifs, an N-capping box and a hydrophobic staple motif, both of which are located between amino acids 151 and 156. The N-capping box consists of a reciprocal hydrogen bonding interaction of Thr152 with Asp155, whereas the hydrophobic staple motif consists of a hydrophobic interaction between Phe151 and Ala156. By contrast with other GSTs, PmGST B1-1 displays distinct hydrogen bond interactions in the N-capping box. In mammalian GSTs these structural elements are critical for protein folding and stability. To investigate the role played by these two motifs in a distantly related organism on the evolutionary scale, site-directed mutagenesis was used to generate several mutants of both motifs in PmGST B1-1. All mutants were efficiently overexpressed and purified, but they were quite unstable, although at different levels, indicating that protein folding was significantly destabilized. The analysis of the T152A and D155G variants indicated that the N-capping box motif plays an important role in the stability and correct folding of the enzyme. The analysis of F151A and A156G mutants revealed that the hydrophobic staple motif influences the structural maintenance of the protein and is implicated in the folding process of PmGST B1-1. Finally, the replacement of Thr152 and Asp155, as well as Phe151 and Ala156 residues influences the catalytic efficiency of the enzyme.

Effect of anaerobic environment on the glutathione transferase isoenzymatic pattern in Proteus mirabilis.

When Proteus mirabilis was cultured anaerobically in the presence of nitrate as terminal electron acceptor, a dramatic reduction of glutathione transferase production occurred. The analysis of the glutathione affinity purified materials in terms of substrate specificity, SDS-PAGE pattern, IEF pattern and immunoblotting revealed that a significantly different glutathione transferase pattern also occurred: two new glutathione transferase forms with an isoelectric point at pH 4.8 and 5.0 appeared. Their N-terminal amino acid sequence analysis as well as the ability to bind to a glutathione affinity column indicate that major differences between anaerobic and aerobic glutathione transferase forms are mainly located in the C-terminal region of the primary structure. In contrast, no significant changes occurred in the production of glutathione transferase isoenzymes when P. mirabilis was grown anaerobically in the absence of a terminal electron acceptor. These results support the idea that bacterial glutathione transferase expression is not strictly related to the absence of oxygen stress.

Characterization of a cDNA encoding metallothionein 3 from cotton (Gossypium hirsutumL.)

A cDNA encoding metallothionein (MT) was isolated from a library constructed with poly A(+) RNA purified from 48 h etiolated cotton (Gossypium hirsutum L.) cotyledons. This cDNA encodes a deduced protein with 63 residues and a molecular weight of 6.3 kDa. The protein has 10 cysteines of which 4 are within the CXXCXCXXXXXC amino-terminus motif and six are within the CXCXXXCXCXXCXC carboxyl-terminus motif characteristic of the type III MT (MT3). The cotton MT3 protein sequence is 76.2, 69.8, 66.7, 60.3 and 33.5% identical to MT3 from Carica papaya, Rubus idaeus, Ribes nigrum, Citrus unshiu, and Gossypium hirsutum type I MT, respectively. A fusion protein was constructed by producing PCR primers for the 5' and 3' ends of the cotton MT3 cDNA and ligating the PCR product inframe at the 3' end of a bacterial glutathione S-transferase (GST) gene in the pGEX3 vector. The 5' PCR primer incorporated a segment of the cotton MT3 noncoding region, resulting in an addition of 9 residues to the MT3 (after Factor Xa digestion site) which increased the size of the expressed protein to 72 residues and 7.6 kDa. Expression of the 7.6 kDa protein in bacteria was confirmed by SDS-PAGE. Induction and accumulation of the GST-MT3 protein began inhibiting bacterial growth after 1 h. Addition of Cu (1 muM to 1 mM), 1 mM cysteine, or 1 mM cystine to the media did not rescue growth. Additionally, this protein was evaluated for its ability to bind Cd, Cu, Ni and Zn in the bacterial expression system. We found that cotton MT3 preferentially binds Cu.

Glutathione-dependent biotransformation of the fungicide chlorothalonil.

A gene responsible for the chlorothalonil biotransformation was cloned from the chromosomal DNA of Ochrobactrum anthropi SH35B, capable of efficiently dissipating the chlorothalonil. The gene encoding glutathione S-transferase (GST) of O. anthropi SH35B was expressed in Escherichia coli, and the GST was subsequently purified by affinity chromatography. The fungicide chlorothalonil was rapidly transformed by the GST in the presence of glutathione. LC-MS analysis supported the formation of mono-, di-, and triglutathione conjugates of chlorothalonil by the GST. The monoglutathione conjugate was observed as an intermediate in the enzymatic reaction. The triglutathione conjugate has not been previously reported and seems to be the final metabolite in the biotransformation of chlorothalonil. The glutathione-dependent biotransformation of chlorothalonil catalyzed by the bacterial GST is reported.

Endocytosis of hepatic lipase and lipoprotein lipase into rat liver hepatocytes in vivo is mediated by the low density lipoprotein receptor-related protein.

In isolated cell studies, the internalization and degradation of hepatic lipase (HL) has been linked to its binding to the low density lipoprotein receptor-related protein (LRP). We have utilized the receptor-associated protein (RAP), a universal inhibitor of high affinity ligand binding to LRP, to evaluate the participation of LRP in the endocytosis of HL and lipoprotein lipase (LPL). We isolated a total endosome fraction from rat livers after a 30-min infusion of recombinant RAP, administered as a glutathione S-transferase conjugate (GST-RAP). GST-RAP infusion had no effect on the concentration of HL in liver homogenates, but its concentration in blood plasma increased progressively by 20%, and enrichment over homogenate of HL in endosomes was reduced by 50% as compared with infusion of GST alone. The concentrations of LPL in liver and plasma were 1.4 and 0.5%, respectively, those of HL, but endosomal enrichment of the two enzymes was similar ( approximately 10-fold). GST-RAP infusion had no effect on the concentration of LPL in liver but increased its concentration in blood plasma by 250% and reduced its endosomal enrichment by 95% or greater. GST-RAP infusion also reduced endosomal enrichment of LRP by 40%, but enrichment of several other endocytic receptors was unaffected. Endosomal enrichment of several membrane trafficking proteins associated with the endocytic pathway in hepatocytes was unaffected by GST-RAP with the exception of early endosome endosome antigen 1, which was reduced by 85%. We conclude that HL is partially and LPL almost exclusively taken up into rat hepatocytes after binding to the endocytic receptor LRP.

GSTB1-1 from Proteus mirabilis: A SNAPSHOT OF AN ENZYME IN THE EVOLUTIONARY PATHWAY FROM A REDOX ENZYME TO A CONJUGATING ENZYME

The native form of the bacterial glutathione transferase B1-1 (EC ) is characterized by one glutathione (GSH) molecule covalently linked to Cys-10. This peculiar disulfide, only found in the Beta and Omega class glutathione S-transferases (GSTs) but absent in all other GSTs, prompts questions about its role and how GSH can be activated and utilized in the reaction normally performed by GSTs. Stopped-flow and spectroscopic experiments suggest that, in the native enzyme (GSTB1-1ox), a second GSH molecule is present, albeit transiently, in the active site. This second GSH binds to the enzyme through a bimolecular interaction followed by a fast thiol-disulfide exchange with the covalently bound GSH. The apparent pK(a) of the non-covalently bound GSH is lowered from 9.0 to 6.4 +/- 0.2 in similar fashion to other GSTs. The reduced form of GSTB1-1 (GSTB1-1red) binds GSH 100-fold faster and also induces a more active deprotonation of the substrate with an apparent pK(a) of 5.2 +/- 0.1. Apparently, the absence of the mixed disulfide does not affect k(cat) and K(m) values in the GST conjugation activity, which is rate-limited by the chemical step both in GSTB1-1red and in GSTB1-1ox. However, GSTB1-1ox follows a steady-state random sequential mechanism whereas a rapid-equilibrium random sequential mechanism is adopted by GSTB1-1red. Remarkably, GSTB1-1ox and GSTB1-1red are equally able to catalyze a glutaredoxin-like catalysis using cysteine S-sulfate and hydroxyethyl disulfide as substrates. Cys-10 is an essential residue in this redox activity, and its replacement by alanine abolishes this enzymatic activity completely. It appears that GSTB1-1 behaves like an "intermediate enzyme" between the thiol-disulfide oxidoreductase and the GST superfamilies.