Evolution of enzymatic activities in the enolase superfamily: crystal structures of the L-Ala-D/L-Glu epimerases from Escherichia coli and Bacillus subtilis.

The members of the enolase superfamily catalyze different overall reactions, yet share a partial reaction that involves Mg(2+)-assisted enolization of the substrate carboxylate anion. The fate of the resulting enolate intermediate is determined by the active site of each enzyme. Several members of this superfamily have been structurally characterized to permit an understanding of the evolutionary strategy for using a common structural motif to catalyze different overall reactions. In the preceding paper, two new members of the superfamily were identified that catalyze the epimerization of the glutamate residue in L-Ala-D/L-Glu. These enzymes belong to the muconate lactonizing enzyme subgroup of the enolase superfamily, and their sequences are only 31% identical. The structure of YcjG, the epimerase from Escherichia coli, was determined by MAD phasing using both the SeMet-labeled protein and a heavy atom derivative. The structure of YkfB, the epimerase from Bacillus subtilis, was determined by molecular replacement using the muconate lactonizing enzyme as a search model. In this paper, we report the three-dimensional structures of these enzymes and compare them to the structure of o-succinylbenzoate synthase, another member of the muconate lactonizing enzyme subgroup.

Evolution of enzymatic activities in the enolase superfamily: identification of the general acid catalyst in the active site of D-glucarate dehydratase from Escherichia coli.

D-Glucarate dehydratase from Escherichia coli (GlucD), a member of the enolase superfamily, catalyzes the dehydration of both D-glucarate and L-idarate to form 5-keto-4-deoxy-D-glucarate (KDG). Previous mutagenesis and structural studies identified Lys 207 and the His 339-Asp 313 dyad as the general basic catalysts that abstract the C5 proton from L-idarate and D-glucarate, respectively, thereby initiating the reaction by formation of a stabilized enediolate anion intermediate [Gulick, A. M., Hubbard, B. K., Gerlt, J. A., and Rayment, I. (2000) Biochemistry 39, 4590-4602]. The vinylogous elimination of the 4-OH group from this intermediate presumably requires a general acid catalyst. The structure of GlucD with KDG and 4-deoxy-D-glucarate bound in the active site revealed that only His 339 and Asn 341 are proximal to the presumed position of the 4-OH leaving group. The N341D and N341L mutants of GlucD were constructed and subjected to both mechanistic and structural analyses. The N341L but not N341D mutant catalyzed the dehydrofluorination of 4-deoxy-4-fluoro-D-glucarate, demonstrating that in this mutant the initial proton abstraction from C5 can be decoupled from elimination of the leaving group from C4. The kinetic properties and structures of these mutants suggest that either Asn 341 participates in catalysis as the general acid that facilitates the departure of the 4-leaving group or is essential for proper positioning of His 339. In the latter scenario, His 339 would function not only as the general base that abstracts the C5 proton from D-glucarate but also as the general acid that catalyzes both the departure of the 4-OH group and the stereospecific incorporation of solvent hydrogen with retention of configuration to form the KDG product. The involvement of a single functional group in this reaction highlights the plasticity of the active site design in members of the enolase superfamily.

Evolution of enzymatic activities in the enolase superfamily: crystallographic and mutagenesis studies of the reaction catalyzed by D-glucarate dehydratase from Escherichia coli.

D-Glucarate dehydratase (GlucD) from Escherichia coli catalyzes the dehydration of both D-glucarate and L-idarate as well as their interconversion via epimerization. GlucD is a member of the mandelate racemase (MR) subgroup of the enolase superfamily, the members of which catalyze reactions that are initiated by abstraction of the alpha-proton of a carboxylate anion substrate. Alignment of the sequence of GlucD with that of MR reveals a conserved Lys-X-Lys motif and a His-Asp dyad homologous to the S- and R-specific bases in the active site of MR. Crystals of GlucD have been obtained into which the substrate D-glucarate and two competitive inhibitors, 4-deoxy-D-glucarate and xylarohydroxamate, could be diffused; D-glucarate is converted to the dehydration product, 5-keto-4-deoxy-D-glucarate (KDG). The structures of these complexes have been determined and reveal the identities of the ligands for the required Mg(2+) (Asp(235), Glu(266), and Asn(289)) as well as confirm the expected presence of Lys(207) and His(339), the catalytic bases that are properly positioned to abstract the proton from C5 of L-idarate and D-glucarate, respectively. Surprisingly, the C6 carboxylate group of KDG is a bidentate ligand to the Mg(2+), with the resulting geometry of the bound KDG suggesting that stereochemical roles of Lys(207) and His(339) are reversed from the predictions made on the basis of the established structure-function relationships for the MR-catalyzed reaction. The catalytic roles of these residues have been examined by characterization of mutant enzymes, although we were unable to use these to demonstrate the catalytic independence of Lys(207) and His(339) as was possible for the homologous Lys(166) and His(297) in the MR-catalyzed reaction.

X-ray structures of the Dictyostelium discoideum myosin motor domain with six non-nucleotide analogs.

The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II complexed with dinitrophenylaminoethyl-, dinitrophenylaminopropyl-, o-nitrophenylaminoethyl-, m-nitrophenylaminoethyl-, p-nitrophenylaminoethyl-, and o-nitrophenyl-N-methyl-aminoethyl-diphosphate.beryllium fluoride have been determined to better than 2.3-A resolution. The structure of the protein and nucleotide binding pocket in these complexes is very similar to that of S1dC.ADP.BeF(x) (Fisher, A. J., Smith, C. A., Thoden, J., Smith, R., Sutoh, K., Holden, H. M., and Rayment, I. (1995) Biochemistry 34, 8960-8972). The position of the triphosphate-like moiety is essentially identical in all complexes. Furthermore, the alkyl-amino group plays the same role as the ribose by linking the triphosphate to the adenine binding pocket; however, none of the phenyl groups lie in the same position as adenine in S1dC.MgADP.BeF(x), even though several of these nucleotide analogs are functionally equivalent to ATP. Rather the former location of adenine is occupied by water in the nanolog complexes, and the phenyl groups are organized in a manner that attempts to optimize their hydrogen bonding interactions with this constellation of solvent molecules. A comparison of the kinetic and structural properties of the nanologs relative to ATP suggests that the ability of a substrate to sustain tension and to generate movement correlates with a well defined interaction with the active site water structure observed in S1dC.MgADP.BeF(x).

Evolution of enzymatic activities in the enolase superfamily: crystal structure of (D)-glucarate dehydratase from Pseudomonas putida.

The structure of (D)-glucarate dehydratase from Pseudomonas putida (GlucD) has been solved at 2.3 A resolution by multiple isomorphous replacement and refined to a final R-factor of 19.0%. The protein crystallizes in the space group I222 with one subunit in the asymmetric unit. The unit cell dimensions are a = 69.6 A, b = 108.8 A, and c = 122.6 A. The crystals were grown using the batch method where the primary precipitant was poly(ethylene glycol) 1000. The structure reveals that GlucD is a tetramer of four identical polypeptides, each containing 451 residues. The structure was determined without a bound substrate or substrate analogue. Three disordered regions are noted: the N-terminus through residue 11, a loop containing residues 99 through 110, and the C-terminus from residue 423. On the basis of primary sequence alignments, we previously concluded that GlucD is a member of the mandelate racemase (MR) subfamily of the enolase superfamily [Babbitt, P. C., Hasson, M. S., Wedekind, J. E., Palmer, D. R. J., Barrett, W. C., Reed, G. J., Rayment, I., Ringe, D., Kenyon, G. L., and Gerlt, J. A. (1996) Biochemistry 35, 16489-16501]. This prediction is now verified, since the overall fold of GlucD is strikingly similar to those of MR, muconate lactonizing enzyme I, and enolase. Also, many of the active site residues of GlucD can be superimposed on those found in the active site of MR. The implications of this structure on the evolution of catalysis in the enolase superfamily are discussed.

X-ray crystal structure of the yeast Kar3 motor domain complexed with Mg.ADP to 2.3 A resolution.

The kinesin family of motor proteins, which contain a conserved motor domain of approximately 350 amino acids, generate movement against microtubules. Over 90 members of this family have been identified, including motors that move toward the minus or plus end of microtubules. The Kar3 protein from Saccharomyces cerevisiae is a minus end-directed kinesin family member that is involved in both nuclear fusion, or karyogamy, and mitosis. The Kar3 protein is 729 residues in length with the motor domain located in the C-terminal 347 residues. Recently, the three-dimensional structures of two kinesin family members have been reported. These structures include the motor domains of the plus end-directed kinesin heavy chain [Kull, F. J., et al. (1996) Nature 380, 550-555] and the minus end-directed Ncd [Sablin, E. P., et al. (1996) Nature 380, 555-559]. We now report the structure of the Kar3 protein complexed with Mg.ADP obtained from crystallographic data to 2.3 A. The structure is similar to those of the earlier kinesin family members, but shows differences as well, most notably in the length of helix alpha 4, a helix which is believed to be involved in conformational changes during the hydrolysis cycle.

X-ray structures of the MgADP, MgATPgammaS, and MgAMPPNP complexes of the Dictyostelium discoideum myosin motor domain.

The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II (S1dC) complexed with MgAMPPNP, MgATPgammaS, and MgADP are reported at 2.1, 1.9, and 2.1 A resolution, respectively. Crystals were obtained by cocrystallization and were isomorphous with respect to those of S1dC. MgADP.BeFx [Fisher, A. J., et al. (1995) Biochemistry 34, 8960-8972]. In all three structures, the electron density for the entire nucleotide was clearly discernible. The overall structures of all three complexes are very similar to that of the beryllium fluoride complex which suggests that the differences in the physiological effects of ATPgammaS and AMPPNP are due to the changes in the equilibrium between the actin-bound and actin-free states of myosin caused by the lower affinity of AMPPNP for myosin. In S1dC.MgAMPPNP, the presence of the bridging nitrogen prompts the side chain of Asn233 to rotate which disrupts the hydrogen bonding pattern in the nucleotide binding pocket and alters the water structure surrounding the ribose hydroxyl groups. It appears that this change is responsible for the reduced affinity of AMPPNP for myosin relative to ATPgammaS. In contrast to the G-proteins, there is no major change in the conformation of the ligands that coordinate the nucleotide in S1dC.MgADP. This is due to three water molecules that adopt the approximate positions of the three oxygens on the gamma-phosphate and maintain the interactions with the Mg2+ ion and protein molecule. Interestingly, the thiophosphate group is evident in S1dC.MgATPgammaS even though it is slowly hydrolyzed by myosin. This suggests that the conformation observed here and in chicken skeletal myosin subfragment-1 [Rayment, I., et al. (1993) Science 261, 50-58] is unable to hydrolyze ATP and represents the structure of the prehydrolysis weak binding state of myosin.

Mechanistic roles of tyrosine 149 and serine 124 in UDP-galactose 4-epimerase from Escherichia coli.

Synthesis and overexpression of a gene encoding Escherichia coli UDP-galactose 4-epimerase and engineered to facilitate cassette mutagenesis are described. General acid-base catalysis at the active site of this epimerase has been studied by kinetic and spectroscopic analysis of the wild-type enzyme and its specifically mutated forms Y149F, S124A, S124V, and S124T. The X-ray crystal structure of Y149F as its abortive complex with UDP-glucose is structurally similar to that of the corresponding wild-type complex, except for the absence of the phenolic oxygen of Tyr 149. The major effects of mutations are expressed in the values of kcat and kcat/Km. The least active mutant is Y149F, for which the value of kcat is 0.010% of that of the wild-type epimerase. The activity of S124A is also very low, with a kcat value that is 0.035% of that of the native enzyme. The values of Km for Y149F and S124A are 12 and 21% of that of the wild-type enzyme, respectively. The value of kcat for S124T is about 30% of that of the wild-type enzyme, and the value of Km is similar to that of the native enzyme. The reactivities of the mutants in UMP-dependent reductive inactivation by glucose are similarly affected, with kobs being decreased by 6560-, 370-, and 3.4-fold for Y149F, S124A, and S124T, respectively. The second-order rate constants for reductive inactivation by NaBH3CN, which does not require general base catalysis, are similar to that for the native enzyme in the cases of S124A, S124T, and S124V. However, Y149F reacts with NaBH3CN 12-20-fold faster than the wild-type enzyme at pH 8.5 and 7.0, respectively. The increased rate for Y149F is attributed to the weakened charge-transfer interaction between Phe 149 and NAD+, which is present with Tyr 149 in the wild-type enzyme. The charge-transfer band is present in the serine mutants, and its intensity at 320 nm is pH-dependent. The pH dependencies of A320 showed that the pKa values for Tyr 149 are 6.08 for the wild-type epimerase, 6.71 for S124A, 6.86 for S124V, and 6.28 for S124T. The low pKa value for Tyr 149 is attributed mainly to the positive electrostatic field created by NAD+ and Lys 153 (4.5 kcal mol-1) and partly to hydrogen bonding with Ser 124 (1 kcal mol-1). The pKa of Tyr 149 is the same as the kinetic pKa for the Bronsted base that facilitates hydride transfer to NAD+. We concluded that Tyr 149 provides the driving force for general acid-base catalysis, with Ser 124 playing an important role in mediating proton transfer.

Molecular structures of the S124A, S124T, and S124V site-directed mutants of UDP-galactose 4-epimerase from Escherichia coli.

UDP-galactose 4-epimerase plays a critical role in sugar metabolism by catalyzing the interconversion of UDP-galactose and UDP-glucose. Originally, it was assumed that the enzyme contained a "traditional" catalytic base that served to abstract a proton from the 4'-hydroxyl group of the UDP-glucose or UDP-galactose substrates during the course of the reaction. However, recent high-resolution X-ray crystallographic analyses of the protein from Escherichia coli have demonstrated the lack of an aspartate, a glutamate, or a histidine residue properly oriented within the active site cleft for serving such a functional role. Rather, the X-ray crystallographic investigation of the epimerase.NADH.UDP-glucose abortive complex from this laboratory has shown that both Ser 124 and Tyr 149 are located within hydrogen bonding distance to the 4'- and 3'-hydroxyl groups of the sugar, respectively. To test the structural role of Ser 124 in the reaction mechanism of epimerase, three site-directed mutant proteins, namely S124A, S124T, and S124V, were constructed and crystals of the S124A.NADH.UDP, S124A.NADH.UDP-glucose, S124T. NADH.UDP-glucose, and S124V.NADH.UDP-glucose complexes were grown. All of the crystals employed in this investigation belonged to the space group P3221 with the following unit cell dimensions: a = b = 83.8 A, c = 108.4 A, and one subunit per asymmetric unit. X-ray data sets were collected to at least 2.15 A resolution, and each protein model was subsequently refined to an R value of lower than 19.0% for all measured X-ray data. The investigations described here demonstrate that the decreases in enzymatic activities observed for these mutant proteins are due to the loss of a properly positioned hydroxyl group at position 124 and not to major tertiary and quaternary structural perturbations. In addition, these structures demonstrate the importance of a hydroxyl group at position 124 in stabilizing the anti conformation of the nicotinamide ring as observed in the previous structural analysis of the epimerase.NADH. UDP complex.

Structural studies on myosin II: communication between distant protein domains.

Understanding how chemical energy is converted into directed movement is a fundamental problem in biology. In higher organisms this is accomplished through the hydrolysis of ATP by three families of motor proteins: myosin, dynein and kinesin. The most abundant of these is myosin, which operates against actin and plays a central role in muscle contraction. As summarized here, great progress has been made towards understanding the molecular basis of movement through the determination of the three-dimensional structures of myosin and actin and through the establishment of systems for site-directed mutagenesis of this motor protein. It now appears that the generation of movement is coupled to ATP hydrolysis by a series of domain movements within myosin.

High-level production and purification of biologically active proteins from bacterial and mammalian cells using the tandem pGFLEX expression system.

Because of the complexities involved in the regulation of gene expression in Escherichia coli and mammalian cells, it is considered general practice to use different vectors for heterologous expression of recombinant proteins in these host systems. However, we have developed and report a shuttle vector system, pGFLEX, that provides high-level expression of recombinant glutathione S-transferase (GST) fusion proteins in E. coli and mammalian cells. pGFLEX contains the cytomegaloma virus (CMV) immediate-early promoter in tandem with the E. coli lacZpo system. The sequences involved in gene expression have been appropriately modified to enable high-level production of fusion proteins in either cell type. The pGFLEX expression system allows production of target proteins fused to either the N or C terminus of the GST pi protein and provides rapid purification of target proteins as either GST fusions or native proteins after cleavage with thrombin. The utility of this vector in identifying and purifying a component of a multi-protein complex is demonstrated with cyclin A. The pGFLEX expression system provides a singular and widely applicable tool for laboratory or industrial production of biologically active recombinant proteins in E. coli and mammalian cells.

Forced evolution of glutathione S-transferase to create a more efficient drug detoxication enzyme.

Glutathione S-transferases (EC 2.5.1.18) in mammalian cells catalyze the conjugation, and thus, the detoxication of a structurally diverse group of electrophilic environmental carcinogens and alkylating drugs, including the antineoplastic nitrogen mustards. We proposed that structural alteration of the nonspecific electrophile-binding site would produce mutant enzymes with increased efficiency for detoxication of a single drug and that these mutants could serve as useful somatic transgenes to protect healthy human cells against single alkylating agents used in cancer chemotherapy protocols. Random mutagenesis of three regions (residues 9-14, 102-112, and 210-220), which together compose the glutathione S-transferase electrophile-binding site, followed by selection of Escherichia coli expressing the enzyme library with the nitrogen mustard mechlorethamine (20-500 microM), yielded mutant enzymes that showed significant improvement in catalytic efficiency for mechlorethamine conjugation (up to 15-fold increase in kcat and up to 6-fold increase in kcat/Km) and that confer up to 31-fold resistance, which is 9-fold greater drug resistance than that conferred by the wild-type enzyme. The results suggest a general strategy for modification of drug- and carcinogen-metabolizing enzymes to achieve desired resistance in both prokaryotic and eukaryotic plant and animal cells.

Mammalian glutathione S-transferase: regulation of an enzyme system to achieve chemotherapeutic efficacy.

The glutathione S-transferases are a family of Phase II detoxication enzymes that catalyze the conjugation of glutathione to a large variety of electrophilic compounds. In the 1990s, there have been many advances regarding the function of these enzymes in protecting a cell from the toxic effects of these electrophiles. The complexity of this enzyme family has been realized and much work has been performed to identify the specific roles played by individual isozymes in resistance to a variety of agents. Likewise, the determination of the crystal structure of these enzymes has allowed the identification of specific amino acid residues that are involved in the catalysis of important reactions. The important role that these enzymes play in carcinogenesis and in drug resistance has warranted an attempt to bring together these different subfields of glutathione S-transferase biology to investigate possible ways that this system could be regulated in therapeutically useful ways. In this report, we have reviewed the recent advances and ways in which this knowledge could be utilized in the advancement of the treatment of cancer.

Structural studies on human glutathione S-transferase pi. Substitution mutations to determine amino acids necessary for binding glutathione.

In order to identify amino acids involved in binding the co-substrate glutathione to the human glutathione S-transferase (GST) pi enzyme, we assembled three criteria to implicate amino acids whose role in binding and catalysis could be tested. Presence of a residue in the highly conserved exon 4 of the GST gene, positional conservation of a residue in 12 glutathione S-transferase amino acid sequences, and results from published chemical modification studies were used to implicate 14 residues. A bacterial expression vector (pUC120 pi), which enabled abundant production (2-26% of soluble Escherichia coli protein) of wild-type or mutant GST pi, was constructed, and, following nonconservative substitution mutation of the 14 implicated residues, five mutants (R13S, D57K, Q64R, I68Y, L72F) showed a greater than 95% decrease in specific activity. A quantitative assay was developed which rapidly measured the ability of wild-type or mutant glutathione S-transferase to bind to glutathione-agarose. Using this assay, each of the five loss of function mutants showed a greater than 20-fold decrease in binding glutathione, an observation consistent with a recent crystal structure analysis showing that several of these residues help to form the glutathione-binding cleft.

Structural studies on human glutathione S-transferase pi. Family of native-specific monoclonal antibodies used to block catalysis.

The glutathione S-transferases are a family of related detoxification enzymes that have been shown to conjugate numerous electrophiles to the common cellular thiol glutathione. We have generated a panel of monoclonal antibodies against the human pi class isozyme of this enzyme, and, in this report, we characterize the binding of these antibodies to the glutathione S-transferase antigen. Of the 10 monoclonal antibodies that we have isolated, 7 are able to recognize the native form of the enzyme while the remaining 3 are only able to bind to glutathione S-transferase pi in assays that partially denature the antigen, such as an enzyme-linked immunosorbent assay or a Western blot. We synthesized seven partial protein fragments and asked whether the monoclonal antibodies could bind to these fragments in an immunoprecipitation reaction. The antibodies that can bind the native form of the enzyme all bind to the carboxyl-terminal domain of the protein. Two antibodies are able to inhibit the glutathione S-transferase-catalyzed reaction noncompetitively against glutathione. Incubation of a 10-fold molar excess of either antibody over enzyme can inhibit the reaction by 50%. We have also used the same protein fragments of glutathione S-transferase pi to show that amino acids 1-77 retain the capacity to bind glutathione in a glutathione-agarose binding assay.

Mutational substitution of residues implicated by crystal structure in binding the substrate glutathione to human glutathione S-transferase pi.

Site-directed substitution mutations were introduced into a cDNA expression vector (pUC120 pi) that encoded a human glutathione S-transferase pi isozyme to non-conservatively replace four residues (Tyr7, Arg13, Gln62 and Asp96). Our earlier X-ray crystallographic analysis implicated these residues in binding and/or chemically activating the substrate glutathione. Each substitution mutation decreased the specific activity of the enzyme to less than 2% of the wild-type. Glutathione-binding was also reduced; however, the Tyr7----Phe mutant still retained 27% of the wild-type capacity to bind glutathione, underlining the primary role that this residue is likely to play in chemically activating the glutathione molecule during catalysis.

Expression of tandem glutathione S-transferase recombinant genes in COS cells for analysis of efficiency of protein expression and associated drug resistance.

Expression vectors were designed and constructed to achieve optimum production of two different isozymes of rat glutathione S-transferase (GST) (EC 2.5.1.18) in COS cells, for studies of drug resistance. Promoter-enhancer elements from the simian virus 40 (SV40) early-region or the mouse alpha 2(I)-collagen gene, GST cDNAs encoding the rat Ya or Yb1 isozymes, and an SV40 replicative origin (ori) were positioned in the vector to express two GSTs at high levels in the same cell. The optimized construct yielded levels of both GST proteins (1% of postmitochondrial protein fraction) that were up to 1.3-fold greater than the sum of those produced individually by two single-unit expression constructs. The best production of the tandem recombinant gene products was observed when the genes were placed in a head to head orientation in close proximity (1 kilobase). With the recombinant genes configured in this way, the plasmid DNA was also amplified in COS cells to higher levels (30% increase over single-unit expression constructs), as ori elements were placed on both DNA strands. Cells expressing the recombinant GSTs were viably sorted by flow cytometry on the basis of a GST-catalyzed conjugation of glutathione to monochlorobimane. Sorted COS cells that expressed both GST Ya and Yb1 from recombinant genes in a tandem, head to head configuration were 25 or 70% more resistant to the alkylating agent chlorambucil than cells that expressed GST Ya or Yb1 alone.