Papers by Bengt Mannervik
Acta Crystallographica Section D Biological Crystallography, 2015
Protein expression and purification, 1996
An expression clone for large-scale production of the polymorphic human glutathione transferase (... more An expression clone for large-scale production of the polymorphic human glutathione transferase (GST) M1-1 has been developed. Heterologous expression in Escherichia coli afforded a yield of 100 mg of GST M1-1 per 3 liters of culture medium, corresponding to an approximately 10-fold increased yield compared to the parental expression construct. Overproduction of the enzyme was dependent on the codon usage in the 5' region of the DNA sequence encoding glutathione transferase M1-1. High-level expression clones were generated by a combination of random silent mutations in selected wobble positions in the coding sequence and immunoselection of clones from the library of random mutants. The strategy used is generally applicable for the production of recombinant proteins provided that a suitable selection procedure is available for identifying the desired mutants.
Protein Science, 2008
Two human Mu class glutathione transferases, hGST M1-1 and hGST M2-2, with high sequence identity... more Two human Mu class glutathione transferases, hGST M1-1 and hGST M2-2, with high sequence identity~84%! exhibit a 100-fold difference in activities with the substrates aminochrome, 2-cyano-1,3-dimethyl-1-nitrosoguanidinẽ cyanoDMNG!, and 1,2-dichloro-4-nitrobenzene~DCNB!, hGST M2-2 being more efficient. A sequence alignment with the rat Mu class GST M3-3, an enzyme also showing high activities with aminochrome and DCNB, demonstrated an identical structural cluster of residues 164-168 in the a6-helices of rGST M3-3 and hGST M2-2, a motif unique among known sequences of human, rat, and mouse Mu class GSTs. A putative electrostatic network Arg107-Asp161-Arg165-Glu164~-Gln167! was identified based on the published three-dimensional structure of hGST M2-2. Corresponding variant residues of hGSTM1-1~Leu165, Asp164, and Arg167! as well as the active site residue Ser209 were targeted for point mutations, introducing hGST M2-2 residues to the framework of hGST M1-1, to improve the activities with substrates characteristic of hGST M2-2. In addition, chimeric enzymes composed of hGST M1-1 and hGST M2-2 sequences were analyzed. The activity with 1-chloro-2,4-dinitrobenzene~CDNB! was retained in all mutant enzymes, proving that they were catalytically competent, but none of the point mutations improved the activities with hGST M2-2 characteristic substrates. The chimeric enzymes showed that the structural determinants of these activities reside in domain II and that residue Arg165 in hGST M2-2 appears to be important for the reactions with cyanoDMNG and DCNB. A mutant, which contained all the hGST M2-2 residues of the putative electrostatic network, was still lacking one order of magnitude of the activities with the characteristic substrates of wild-type hGST M2-2. It was concluded that a limited set of point mutations is not sufficient, but that indirect secondary structural affects also contribute to the hGST M2-2 characteristic activities with aminochrome, cyanoDMNG, and DCNB.
Protein Expression and Purification, 1995
Engineering of a metal coordinating site into human glutathione transferase M1-1 based on immobilized metal ion affinity chromatography of homologous rat enzymes
"Protein Engineering, Design and Selection", 1994
Rat glutathione transferase (GST) 3-3 binds to Ni(II)-iminodiacetic acid (IDA)-agarose, whereas o... more Rat glutathione transferase (GST) 3-3 binds to Ni(II)-iminodiacetic acid (IDA)-agarose, whereas other GSTs that are abundant in rat liver do not bind to this immobilized metal ion affinity chromatography (IMAC) adsorbent. Rat GST 3-3 contains two superficially located amino acid residues, His84 and His85, that are suitably positioned for coordination to Ni(II)-IDA-agarose. This particular structural motif is lacking in GSTs that do not bind to the IMAC matrix. Creation of an equivalent His-His structure in the homologous human GST M1-1 by protein engineering afforded a mutant enzyme that displays affinity for Ni(II)-IDA-agarose, in contrast to the wild-type GST M1-1. The results identify a distinct site that is operational in IMAC and suggest an approach to the rational design of novel integral metal coordination sites in proteins.
Protein Engineering Design and Selection, 2007
A library of recombinant glutathione transferases (GSTs) generated by shuffling of DNA encoding h... more A library of recombinant glutathione transferases (GSTs) generated by shuffling of DNA encoding human GST M1-1 and GST M2-2 was screened with eight alternative substrates, and the activities were subjected to multivariate analysis. Assays were made in lysates of bacteria in which the GST variants had been expressed. The primary data showed clustering of the activities in eightdimensional substrate-activity space. For an incisive analysis, the rows of the data matrix, corresponding to the different enzyme variants, were individually scaled to unit length, thus accounting for different expression levels of the enzymes. The columns representing the activities with alternative substrates were subsequently individually normalized to unit variance and a zero mean. By this standardization, the data were adjusted to comparable orders of magnitude. Three molecular quasi-species were recognized by multivariate K-means and principal component analyses. Two of them encompassed the parental GST M1-1 and GST M2-2. A third one diverged functionally by displaying enhanced activities with some substrates and suppressed activities with signature substrates for GST M1-1 and GST M2-2. A fourth cluster contained mutants with impaired functions and was not regarded as a quasi-species. Sequence analysis of representatives of the mutant clusters demonstrated that the majority of the variants in the diverging novel quasi-species were structurally similar to the M1-like GSTs, but distinguished themselves from GST M1-1 by a Ser to Thr substitution in the active site. The data show that multivariate analysis of functional profiles can identify small structural changes influencing the evolution of enzymes with novel substrate-activity profiles.
Detoxication of base propenals and other alpha, beta-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases
Proceedings of the National Academy of Sciences, 1994
Radiation and chemical reactions that give rise to free radicals cause the formation of highly cy... more Radiation and chemical reactions that give rise to free radicals cause the formation of highly cytotoxic base propenals, degradation products of DNA. Human glutathione transferases (GSTs; RX:glutathione R-transferase, EC 2.5.1.18) of classes Alpha, Mu, and Pi were shown to promote the conjugation of glutathione with base propenals and related alkenes. GST P1-1 was particularly active in catalyzing the reactions with the propenal derivatives, and adenine propenal was the substrate giving the highest activity. The catalytic efficiency of GST P1-1 with adenine propenal (kcat/Km = 7.7 x 10(5) M-1.s-1) is the highest so far reported with any substrate for this enzyme. In general, GST A1-1 and GST M1-1, in contrast to GST P1-1, were more active with 4-hydroxyalkenals (products of lipid peroxidation) than with base propenals. The adduct resulting from the Michael addition of glutathione to the alkene function of one of the base propenals (adenine propenal) was identified by mass spectrometry. At the cellular level, GST P1-1 was shown to provide protection against alpha, beta-unsaturated aldehydes. GST P1-1 added to the culture medium of HeLa cells augmented the protective effect of glutathione against the toxicity of adenine propenal and thymine propenal. No protective effect of the enzyme was observed in the presence of the competitive inhibitor S-hexylglutathione. GST P1-1 introduced into Hep G2 cells by electroporation was similarly found to increase their resistance to acrolein. The results show that glutathione transferases may play an important role in cellular detoxication of electrophilic alpha, beta-unsaturated carbonyl compounds produced by radical reactions, lipid peroxidation, ionizing radiation, and drug metabolism.
Functionally diverging molecular quasi-species evolve by crossing two enzymes
Proceedings of the National Academy of Sciences, 2006
Molecular evolution is frequently portrayed by structural relationships, but delineation of separ... more Molecular evolution is frequently portrayed by structural relationships, but delineation of separate functional species is more elusive. We have generated enzyme variants by stochastic recombinations of DNA encoding two homologous detoxication enzymes, human glutathione transferases M1-1 and M2-2, and explored their catalytic versatilities. Sampled mutants were screened for activities with eight alternative substrates, and the activity fingerprints were subjected to principal component analysis. This phenotype characterization clearly identified at least three distributions of substrate selectivity, where one was orthogonal to those of the parent-like distributions. This approach to evolutionary data mining serves to identify emerging molecular quasi-species and indicates potential trajectories available for further protein evolution.
Proceedings of the National Academy of Sciences, 2004
A strategy for rational enzyme design is reported and illustrated by the engineering of a protein... more A strategy for rational enzyme design is reported and illustrated by the engineering of a protein catalyst for thiol-ester hydrolysis. Five mutants of human glutathione (GSH; ␥-Glu-Cys-Gly) transferase A1-1 were designed in the search for a catalyst and to provide a set of proteins from which the reaction mechanism could be elucidated. The single mutant A216H catalyzed the hydrolysis of the S-benzoyl ester of GSH under turnover conditions with a kcat͞KM of 156 M ؊1 ⅐min ؊1 , and a catalytic proficiency of >10 7 M ؊1 when compared with the first-order rate constant of the uncatalyzed reaction. The wild-type enzyme did not hydrolyze the substrate, and thus, the introduction of a single histidine residue transformed the wild-type enzyme into a turnover system for thiol-ester hydrolysis. By kinetic analysis of single, double, and triple mutants, as well as from studies of reaction products, it was established that the enzyme A216H catalyzes the hydrolysis of the thiol-ester substrate by a mechanism that includes an acyl intermediate at the side chain of Y9. Kinetic measurements and the crystal structure of the A216H GSH complex provided compelling evidence that H216 acts as a general-base catalyst. The introduction of a single His residue into human GSH transferase A1-1 created an unprecedented enzymatic function, suggesting a strategy that may be of broad applicability in the design of new enzymes. The protein catalyst has the hallmarks of a native enzyme and is expected to catalyze various hydrolytic, as well as transesterification, reactions.
Catalytic Efficiency of Glutathione Transferase P1-1 Variants Towards Bay- and Fjord-Region Diol Epoxides of Polycyclic Aromatic Hydrocarbons
Polycyclic Aromatic Compounds, 1999
Polymorphism of the human GSTP gene may be of importance in PAH-induced carcinogenesis. A higher ... more Polymorphism of the human GSTP gene may be of importance in PAH-induced carcinogenesis. A higher frequency of GSTP*B (encoding for Val at position 105) genotype among individuals with certain tumours has been observed. The altered susceptibility may be associated with a decreased GST-dependent elimination of reactive PAH-intermediates. We have determined the catalytic efficiency of two naturally occurring GSTP1-1 allelic variants
Inactivation of Carcinogenic Diol Epoxides of Dibenzo[ a,l ]pyrene (Dibenzo[ def,p ]chrysene) by Human Alpha Class Glutathione Transferases
Polycyclic Aromatic Compounds, 2002
Human Alpha class glutathione transferases (hGSTs) have been incubated with the ultimate carcinog... more Human Alpha class glutathione transferases (hGSTs) have been incubated with the ultimate carcinogenic ( m )- anti - and (+)- syn -diol epoxides (DE) of the nonplanar dibenzo[ a,l ]pyrene (DBP). hGSTA1-1, A2-2, and A3-3 demonstrate activity with both diol epoxides ( R -absolute configuration at the benzylic oxirane carbon) whereas hGSTA4-4 virtually was inactive. (+)- syn -DBPDE was superior
Journal of the American Chemical Society, 1998
A major detoxification pathway used by aerobic organisms involves the enzymatic conjugation of th... more A major detoxification pathway used by aerobic organisms involves the enzymatic conjugation of the tripeptide glutathione (GSH) to the electrophilic center of toxic substances by the glutathione transferases (GSTs) (EC 2.5.1.18). 1 These enzymes activate the cysteine thiol group of GSH for nucleophilic addition to a variety of substrates, including aryl halides, R, -unsaturated aldehydes and ketones, and epoxides. However, despite a large number of biochemical and structural studies, 2 the mechanism by which GSH transferases catalyze these addition reactions remains unclear. The three-dimensional structures have shown that the hydroxyl group of an active site tyrosine residue (Tyr9 for human GST A1-1, class alpha), which is conserved among the majority of known GSH transferases, is within hydrogenbonding distance of the sulfur of glutathione. 2 In solution, the pK a of GSH is about 9.0, 3 whereas in the GST A1-1 enzyme-GSH complex the pK a of the thiol group is 6.2. 4 The tyrosine residue of this complex is believed to stabilize the thiolate of GSH through a hydrogen-bonding interaction (TyrOH‚‚‚ -SG). 5 Alternatively, the abnormally low pK a of the tyrosyl hydroxyl group may provide a tyrosinate anion which can act as a general base (TyrO -‚‚‚HSG) 6 to abstract the proton from the sulfhydryl group. To investigate this issue, we have used unnatural amino acid mutagenesis to site-specifically replace Tyr9 in human GST A1-1 with a series of fluorinated tyrosine analogues with pK a values ranging from 5.3 to 10. The observed values of k cat and the pH dependence of the mutants are relatively unaffected by fluorine substitution with the exception of the 2,3,5,6-tetrafluorotyrosine mutant. The results are consistent with a previously proposed hydrogen-bonding role for Tyr9 in human GST A1-1 4,7 and are compared with previous experimental 8 and theoretical studies 9 of GSTs in which the pK a of the active tyrosine residue is altered.
Journal of Molecular Biology, 1995
Arg15 is a conserved active-site residue in class Alpha glutathione transferases. X-ray diffracti... more Arg15 is a conserved active-site residue in class Alpha glutathione transferases. X-ray diffraction studies of human glutathione transferase A1-1 have shown that N' of this amino acid residue is adjacent to the sulfur atom of a glutathione derivative bound to the active site, suggesting the presence of a hydrogen bond. The phenolic hydroxyl group of Tyr9 also forms a hydrogen bond to the sulfur atom of glutathione, and removal of this hydroxyl group causes partial inactivation of the enzyme. The present study demonstrates by use of site-directed mutagenesis the functional significance of Arg15 for catalysis. Mutation of Arg15 into Leu reduced the catalytic activity by 25-fold, whereas substitution by Lys caused only a threefold decrease, indicating the significance of a positively charged residue at position 15. Mutation of Arg15 into Ala or His caused a substantial reduction of the specific activity (200 or 400-fold, respectively), one order of magnitude more pronounced than the effect of the Tyr9~Phe mutation. Double mutations involving residues 9 and 15 demonstrated that the effects of mutations at the two positions were additive except for the substitution of His for Arg15, which appeared to cause secondary structural effects. The pK~ value of the phenolic hydroxyl of Tyr9 was determined by UV absorption difference spectroscopy and was found to be 8.1 in the wild-type enzyme. The corresponding pK~ values of mutants R15K, R15H and R15L were 8.5, 8.7 and 8.8, respectively, demonstrating the contribution of the guanidinium group of Arg15 to the electrostatic field in the active site. Addition of glutathione caused an increased pK, value of Tyr9; this effect was not obtained with S-methylglutathione. These results show that Tyr9 is protonated when glutathione is bound to the enzyme at physiological pH values. The involvement of an Arg residue in the binding and activation of glutathione is a feature that distinguishes class Alpha glutathione transferases from members in other glutathione transferase classes.
Journal of Molecular Biology, 2000
An N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) is strictly conserved at the beginning of helix a6 i... more An N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) is strictly conserved at the beginning of helix a6 in the core of virtually all glutathione transferases (GST) and GST-related proteins. It has been demonstrated that this local motif is important in determining the a-helical propensity of the isolated a6-peptide and plays a crucial role in the folding and stability of GSTs. Its removal by site-directed mutagenesis generated temperature-sensitive folding mutants unable to refold at physiological temperature (37 C). In the present work, variants of human GSTP1-1 (S150A and D153A), in which the capping residues have been substituted by alanine, have been generated and puri®ed for structural analysis. Thus, for the ®rst time, temperature-sensitive folding mutants of an enzyme, expressed at a permissive temperature, have been crystallized and their three-dimensional structures determined by X-ray crystallography. The crystal structures of human pi class GST temperature-sensitive mutants provide a basis for understanding the structural origin of the dramatic effects observed on the overall stability of the enzyme at higher temperatures upon single substitution of a capping residue.
Journal of Molecular Biology, 2000
Hybridization of alpha class subunits generating a functional glutathione transferase A1-4 heterodimer
Journal of Molecular Biology, 2002
Within the Alpha class of the mammalian glutathione transferases two variants of subunit interfac... more Within the Alpha class of the mammalian glutathione transferases two variants of subunit interfaces exist. One is conserved among the A4 subunits, whereas the second one is found in all other members of the Alpha class. The ability of the two Alpha class subunit interfaces to adopt a functional heterodimeric structure has been investigated here.The heterodimer GST A1-4 was obtained by co-expression of the two human Alpha class subunits A1 and A4 in Escherichia coli. A histidine tail was added to the N terminus of the A1 subunit to facilitate the purification of the heterodimer. The heterodimer was formed in a small proportion implying that the efficiency of the hybridization between subunit A1 and A4 is less than the propensity for homodimer formation. The hybrid enzyme was stable at low temperatures, but the two subunits dissociated and reassociated into homodimers at 40 degrees C. Three different substrates were used for subunit-selective kinetic characterization of the GST A1-4 heterodimer: 1-chloro-2,4-dinitrobenzene, nonenal and Delta(5)-androstene-3,17-dione. Both subunit A1 and subunit A4 were active in GST A1-4, but the specific activities and k(cat) values were lower than the average values of the two parental isoenzymes. However, at high temperatures the subunits of the hybrid enzyme dissociated and formed homodimers, and the activities increased to expected values. Hence, the low activities of the individual subunits in the heterodimer were reversible. The non-additive kinetic properties of the subunits in the heterodimer therefore highlight the importance of fine-tuned subunit interactions for optimal catalytic efficiency of GST A1-1 and GST A4-4.
Journal of Molecular Biology, 1999
Here the structure of human glyoxalase II has been investigated by studying unfolding at equilibr... more Here the structure of human glyoxalase II has been investigated by studying unfolding at equilibrium and refolding. Human glyoxalase II contains two tryptophan residues situated at the N-terminal (Trp57) and C-terminal (Trp199) regions of the molecule. Trp57 is a non-conserved residue located within a``zinc binding motif'' (T/SHXHX 57 DH) which is strictly conserved in all known glyoxalase II sequences as well as in metal-dependent b-lactamase and arylsulfatase. Site-directed mutagenesis has been used to construct single-tryptophan mutants in order to characterize better the guanidine-induced unfolding intermediates. The denaturation at equilibrium of wild-type glyoxalase II, as followed by activity, intrinsic¯uorescence and CD, is multiphasic, suggesting that different regions of varying structural stability characterize the native structure of glyoxalase II. At intermediate denaturant concentration (1.2 M guanidine) a molten globule state is attained. The reactivation of the denatured wild-type enzyme occurs only in the presence of Zn(II) ions. The results show that Zn(II) is essential for the maintenance of the native structure of glyoxalase II and that its binding to the apoenzyme occurs during an essential step of refolding. The comparison of unfoldinḡ uorescence transitions of single-trypthophan mutants with that of wildtype enzyme indicates that the strictly conserved``zinc binding motif'' is located in a¯exible region of the active site in which Zn(II) participates in catalysis.
Journal of Molecular Biology, 1999
The oxidation of lipids and cell membranes generates cytotoxic compounds implicated in the etiolo... more The oxidation of lipids and cell membranes generates cytotoxic compounds implicated in the etiology of aging, cancer, atherosclerosis, neurodegenerative diseases, and other illnesses. Glutathione transferase (GST) A4-4 is a key component in the defense against the products of this oxidative stress because, unlike other Alpha class GSTs, GST A4-4 shows high catalytic activity with lipid peroxidation products such as 4-hydroxynon-2-enal (HNE). The crystal structure of human apo GST A4-4 unexpectedly possesses an ordered C-terminal a-helix, despite the absence of any ligand. The structure of human GST A4-4 in complex with the inhibitor S-(2-iodobenzyl) glutathione reveals key features of the electrophilic substrate-binding pocket which confer speci®city toward HNE. Three structural modules form the binding site for electrophilic substrates and thereby govern substrate selectivity: the b1-a1 loop, the end of the a4 helix, and the C-terminal a9 helix. A few residue changes in GST A4-4 result in a9 taking over a predominant role in ligand speci-®city from the N-terminal loop region important for GST A1-1. Thus, the C-terminal helix a9 in GST A4-4 provides pre-existing ligand complementarity rather than acting as a¯exible cap as observed in other GST structures. Hydrophobic residues in the a9 helix, differing from those in the closely related GST A1-1, delineate a hydrophobic speci®city canyon for the binding of lipid peroxidation products. The role of residue Tyr212 as a key catalytic residue, suggested by the crystal structure of the inhibitor complex, is con®rmed by mutagenesis results. Tyr212 is positioned to interact with the aldehyde group of the substrate and polarize it for reaction. Tyr212 also coopts part of the binding cleft ordinarily formed by the N-terminal substrate recognition region in the homologous enzyme GST A1-1 to reveal an evolutionary swapping of function between different recognition elements. A structural model of catalysis is presented based on these results.
Journal of Molecular Biology, 1998
Human glutathione transferase P1-1 (GSTP1-1) is polymorphic in amino acid residue 105, positioned... more Human glutathione transferase P1-1 (GSTP1-1) is polymorphic in amino acid residue 105, positioned in the substrate binding H-site. To elucidate the role of this residue an extensive characterization of GSTP1-1/Ile105 and GSTP1-1/Val105 was performed. Mutant enzymes with altered volume and hydrophobicity of residue 105, GSTP1-1/Ala105 and GSTP1-1/Trp105, were constructed and included in the study. Steady-state kinetic parameters and speci®c activities were determined using a panel of electrophilic substrates, with the aim of covering different types of reaction mechanisms. Analysis of the steady-state kinetic parameters indicates that the effect of the substitution of the amino acid in position 105 is highly dependent on substrate used. When 1-chloro-2,4-dinitrobenzene was used as substrate a change in the side-chain of residue 105 seemed primarily to cause changes in the K M value, while the k cat value was not distinctively affected. With other substrates, such as 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and ethacrynic acid both k cat and K M values were altered by the substitution of amino acid 105. The constant for formation of the s-complex between 1,3,5-trinitrobenzene and glutathione was shown to be dependent upon the volume of the amino acid in position 105. The nature of the amino acid in position 105 was also shown to affect the thermal stability of the enzyme at 50 C, indicating an important role for this residue in the stabilization of the enzyme. The GSTP1-1/ Ile105 variant was approximately two to three times more stable than the Val105 variant as judged by their half-lives. The presence of glutathione in the incubation buffer afforded a threefold increase in the half-lives of the enzymes. Thus, the thermal stability of the enzyme and depending on substrate, both K M values and turnover numbers are in¯uenced by substitutions in position 105 of GSTP1-1.
Journal of Molecular Biology, 1995
Human glutathione transferase A1-1 can be expressed as a fusion protein with coat protein III of ... more Human glutathione transferase A1-1 can be expressed as a fusion protein with coat protein III of filamentous phage f1 in a form that allows selection Uppsala University among variant mutant forms based on specific adsorption to immobilized Biomedical Center, Box 576 active-site ligands. A library of mutant enzymes differing in the active-site S-751 23 Uppsala, Sweden region was generated by random mutagenesis of ten amino acid residues involved in the binding of electrophilic substrates. Novel glutathione transferases with altered specificity for active-site ligands were isolated by adsorption of the fusion protein on the surface of phage to analogs of an electrophilic substrate. Thus, phage display of glutathione transferase affords a system for engineering novel binding specificities onto the pre-existing protein framework of the enzyme.
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Papers by Bengt Mannervik