Emergence of novel enzyme quasi-species depends on the substrate matrix

Current research on enzyme evolution has shown that many enzymes are promiscuous and have activities with alternative substrates. Mutagenesis tends to relax substrate selectivity, and evolving enzymes can be regarded (summed over evolutionary time) as clusters of enzyme variants, or “quasi-species,” tested against a “substrate matrix” defined by all chemical substances to which the evolvants are exposed.In this investigation, the importance of the substrate matrix for identification of evolvable clusters of enzymes was evaluated by random sampling of variants from a library of glutathione transferase (GST) mutants. The variant GSTs were created by DNA shuffling of homologous Alpha class sequences. The substrate matrix was an array of alternative substrates used under defined experimental conditions. The measured enzyme activities produced a rectangular matrix, in which the rows can be projected as enzyme vectors in substrate-activity space and, reciprocally, the columns can be projected as alternative substrate vectors in enzyme-activity space. Multivariate analysis of the catalytic activities demonstrated that the enzyme vectors formed two primary clusters or functional “molecular quasi-species.” These quasi-species serve as the raw material from which more specialized enzymes eventually could evolve. The substrate vectors similarly formed two major groups. Identification of separate quasi-species of GSTs in a mutant library was critically dependent on the nature of the substrate matrix. When substrates from just one of the two groups were used, only one cluster of enzymes could be recognized. On the other hand, expansion of the substrate matrix to include additional substrates showed the presence of a third quasi-species among the GST variants already analyzed. Thus, the portrayal of the functional quasi-species is intimately linked to the effective substrate matrix. In natural evolution, the substrates actually encountered therefore play a pivotal role in determining whether latent catalytic abilities become manifest in novel enzymes.     

Residue 234 is a master switch of the alternative-substrate activity profile of human and rodent theta class glutathione transferase T1-1

Background

The Theta class glutathione transferase GST T1-1 is a ubiquitously occurring detoxication enzyme. The rat and mouse enzymes have high catalytic activities with numerous electrophilic compounds, but the homologous human GST T1-1 has comparatively low activity with the same substrates. A major structural determinant of substrate recognition is the H-site, which binds the electrophile in proximity to the nucleophilic sulfur of the second substrate glutathione. The H-site is formed by several segments of amino acid residues located in separate regions of the primary structure. The C-terminal helix of the protein serves as a lid over the active site, and contributes several residues to the H-site.

Methods

Site-directed mutagenesis of the H-site in GST T1-1 was used to create the mouse Arg234Trp for comparison with the human Trp234Arg mutant and the wild-type rat, mouse, and human enzymes. The kinetic properties were investigated with an array of alternative electrophilic substrates to establish substrate selectivity profiles for the different GST T1-1 variants.

Results

The characteristic activity profile of the rat and mouse enzymes is dependent on Arg in position 234, whereas the human enzyme features Trp. Reciprocal mutations of residue 234 between the rodent and human enzymes transform the substrate-selectivity profiles from one to the other.

Conclusions

H-site residue 234 has a key role in governing the activity and substrate selectivity profile of GST T1-1.

General significance

The functional divergence between human and rodent Theta class GST demonstrates that a single point mutation can enable or suppress enzyme activities with different substrates.     

Emergence of a novel highly specific and catalytically efficient enzyme from a naturally promiscuous glutathione transferase

Redesign of glutathione transferases (GSTs) has led to enzymes with remarkably enhanced catalytic properties. Exchange of substrate-binding residues in GST A1-1 created a GST A4-4 mimic, called GIMFhelix, with >300-fold improved activity with nonenal and suppressed activity with other substrates. In the present investigation GIMFhelix was compared with the naturally-evolved GSTs A1-1 and A4-4 by determining catalytic efficiencies with nine alternative substrates. The enzymes can be represented by vectors in multidimensional substrate-activity space, and the vectors of GIMFhelix and GST A1-1, expressed in kcat/Km values for the alternative substrates, are essentially orthogonal. By contrast, the vectors of GIMFhelix and GST A4-4 have approximately similar lengths and directions. The broad substrate acceptance of GST A1-1 contrasts with the high selectivity of GST A4-4 and GIMFhelix for alkenal substrates. Multivariate analysis demonstrated that among the diverse substrates used, nonenal, cumene hydroperoxide, and androstenedione are major determinants in the portrayal of the three enzyme variants. These GST substrates represent diverse chemistries of naturally occurring substrates undergoing Michael addition, hydroperoxide reduction, and steroid double-bond isomerization, respectively. In terms of function, GIMFhelix is a novel enzyme compared to its progenitor GST A1-1 in spite of 94% amino-acid sequence identity between the enzymes. The redesign of GST A1-1 into GIMFhelix therefore serves as an illustration of divergent evolution leading to novel enzymes by minor structural modifications in the active site. Notwithstanding low sequence identity (60%), GIMFhelix is functionally an isoenzyme of GST A4-4.     

Structural Basis for Featuring of Steroid Isomerase Activity in Alpha Class Glutathione Transferases

Glutathione transferases (GSTs) are abundant enzymes catalyzing the conjugation of hydrophobic toxic substrates with glutathione. In addition to detoxication, human GST A3-3 displays prominent steroid double-bond isomerase activity; e.g. transforming Δ⁵-androstene-3-17-dione into Δ⁴-androstene-3-17-dione (AD). This chemical transformation is a crucial step in the biosynthesis of steroids, such as testosterone and progesterone. In contrast to GST A3-3, the homologous GST A2-2 does not show significant steroid isomerase activity. We have solved the 3D structures of human GSTs A2-2 and A3-3 in complex with AD. In the GST A3-3 crystal structure, AD was bound in an orientation suitable for the glutathione (GSH)-mediated catalysis to occur. In GST A2-2, however, AD was bound in a completely different orientation with its reactive double bond distant from the GSH-binding site. The structures illustrate how a few amino acid substitutions in the active site spectacularly alter the binding mode of the steroid substrate in relation to the conserved catalytic groups and an essentially fixed polypeptide chain conformation. Furthermore, AD did not bind to the GST A2-2-GSH complex. Altogether, these results provide a first-time structural insight into the steroid isomerase activity of any GST and explain the 5000-fold difference in catalytic efficiency between GSTs A2-2 and A3-3. More generally, the structures illustrate how dramatic diversification of functional properties can arise via minimal structural alterations. We suggest a novel structure-based mechanism of the steroid isomerization reaction.     

Single chain antibody displays glutathione S-transferase activity

Substrate binding and the subsequent reaction are the two principal phenomena that underlie the activity of enzymes, and many enzyme-like catalysts were generated based on the phenomena. The single chain variable region fragment of antibody 2F3 (scFv2F3) was elicited against hapten GSH-S-DN2phBu, a conjugate of glutathione (GSH), butyl alcohol, and 1-chloro-2,4-dinitrobenzene (CDNB); it can therefore bind both GSH and CDNB, the substrates of native glutathione S-transferases (GSTs). It was shown previously that there is a serine residue that is the catalytic group of GST in the CDR regions of scFv2F3 close to the sulfhydryl of GSH. Thus, we anticipated that scFv2F3 will display GST activity. The experimental results showed that scFv2F3 indeed displayed GST activity that is equivalent to the rat-class GST T-2-2 and exhibited pH- and temperature-dependent catalytic activity. Steady-state kinetic studies showed that the Km values for the substrates are close to those of native GSTs, indicating that scFv2F3 has strong affinities for the substrates. Compared with some other GSTs, its kcat value was found to be low, which could be caused by the similarity between the GSH-S-DN2phBu and the reaction product of GSH and CDNB. These results showed that our approach to imitating enzymes is correct, which is that an active site may catalyze a chemical reaction when a catalytic group locates beside a substrate-binding site of a receptor. It is important to consider product inhibition in hapten design in order to obtain a mimic with a high catalytic efficiency.     

Role of invariant tyrosines in a crustacean mu-class glutathione S-transferase from shrimp Litopenaeus vannamei: site-directed mutagenesis of Y7 and Y116

Y6 and Y115 are key amino acids involved in enzyme-substrate interactions in mu-class glutathione S-transferase (GST). They provide electrophilic assistance and stabilize substrates through their hydroxyl groups. Two site-directed mutants (Y7F and Y116F) and the wild-type shrimp GSTs were expressed in Escherichia coli, and the steady-state kinetic parameters were determined using CDNB as the second substrate. The mutants were modeled based on a crystal structure of a mu-class GST to obtain further insights about the changes at the active site. The Y116F mutant had an increase in kcat contrary to Y7F compared to the wild type. Molecular modeling showed that the shrimp GST has a H108 residue that may contribute to compensate and lead to a less deleterious change when conserved tyrosine residues are mutated. This work indicates that shrimp GST is a useful model to understand the catalysis mechanisms in this critical enzyme.     

Structural determinants of glutathione transferases with azathioprine activity identified by DNA shuffling of alpha class members

A library of alpha class glutathione transferases (GSTs), composed of chimeric enzymes derived from human (A1-1, A2-2 and A3-3), bovine (A1-1) and rat (A2-2 and A3-3) cDNA sequences was constructed by the method of DNA shuffling. The GST variants were screened in bacterial lysates for activity with the immunosuppressive agent azathioprine, a prodrug that is transformed into its active form, 6-mercaptopurine, by reaction with the tripeptide glutathione catalyzed by GSTs. Important structural determinants for activity with azathioprine were recognized by means of primary structure analysis and activities of purified enzymes chosen from the screening. The amino acid sequences could be divided into 23 exchangeable segments on the basis of the primary structures of 45 chosen clones. Segments 2, 20, 21, and 22 were identified as primary determinants of the azathioprine activity representing two of the regions forming the substrate-binding H-site. Segments 21 and 22 are situated in the C-terminal helix characterizing alpha class GSTs, which is instrumental in their catalytic function. The study demonstrates the power of DNA shuffling in identifying segments of primary structure that are important for catalytic activity with a targeted substrate. GSTs in combination with azathioprine have potential as selectable markers for use in gene therapy. Knowledge of activity-determining segments in the structure is valuable in the protein engineering of glutathione transferase for enhanced or suppressed activity.     

S-(4-Nitrophenacyl)glutathione is a specific substrate for glutathione transferase omega 1-1

Glutathione transferase omega 1-1 (GSTO1-1) catalyzes the biotransformation of arsenic and is implicated as a factor influencing the age-at-onset of Alzheimer’s disease and the posttranslational activation of interleukin 1β (IL-1β). Investigation of the biological role of GSTO1-1 variants has been hampered by the lack of a specific assay for GSTO1-1 activity in tissue samples that contain other GSTs and other enzymes with similar catalytic specificities. Previous studies (P. G. Board and M. W. Anders, Chem. Res. Toxicol. 20 (2007) 149-154) have shown that GSTO1-1 catalyzes the reduction of S-(phenacyl)glutathiones to acetophenones. A new substrate, S-(4-nitrophenacyl)glutathione (4NPG), has been prepared and found to have a high turnover with GSTO1-1 but negligible activity with GSTO2-2 and other members of the glutathione transferase superfamily. A spectrophotometric assay with 4NPG as a substrate has been used to determine GSTO1-1 activity in several human breast cancer cell lines and in mouse liver and brain tissues.     

Developmental studies on the Sigma and Delta-1 glutathione transferases of Lucilia cuprina

The glutathione transferases (GSTs) are a large group of enzymes having both detoxication roles and specialist metabolic functions. The present work represents an initial approach to identifying some of these roles by examining the variation of specific members of the family under differing conditions. The GSTs from Lucilia cuprina have been partially purified, members of two families being isolated, by the use of glutathione immobilised on epichlorhydrin-activated Sepharose 6B. The GSTs were separated by 2D SDS-PAGE and characterised by MALDI-TOF analysis of tryptic peptides. The mass fragments were then matched against the corresponding Drosophila melanogaster and Musca domestica sequences. GSTs were identified as coming from only the Sigma and Delta classes. The multiple Delta zones appear all to be derived from the Lucilia GSTD1 isoform. The distribution of these GST proteins has been studied during different developmental stages of the insect. Delta isoforms were present in all developmental stages of L. cuprina. The Sigma GST was not detectable in the egg, was just detectable in the larval and pupal stages and was the major GST isolated in the adult. Sigma and Delta isoforms were both found in all body segments of the insect. Both isoforms appear to undergo extensive post-translational modification. Activities of the two types of protein with model substrates have been determined.     

Importance of a hypervariable active-site residue in human Mu class glutathione transferases catalyzing the bioactivation of chemotherapeutic thiopurine prodrugs

Glutathione transferases (GSTs) catalyze the bioactivation of the thiopurine prodrugs azathioprine, cis-6-(2-acetylvinylthio)purine (cAVTP) and trans-6-(2-acetylvinylthio)guanine (tAVTG), thereby releasing the antimetabolites 6-mercaptopurine and 6-thioguanine. In the GST Mu class, GST M1-1 has the highest catalytic efficiency, whereas GST M2-2 and other enzymes are less active. In the evolution of Mu class GSTs, residue 210 appears hypervariable and has particular functional significance. We demonstrate that the catalytic activity of GST M1-1 with cAVTP or tAVTG is successively diminished when wild-type Ser-210 is mutated into Ala followed by Thr. Conversely, mutating wild-type Thr-210 in GST M2-2 into Ala and Ser enhanced the corresponding activities. Comparisons were also made with GST M2-2 distinguished by Gly or Pro in position 210, as well as wild-type GSTs M4-4 and M5-5. The results suggest that the hydroxyl group of Ser in position 210 stabilizes the transition state of the GST-catalyzed reaction. The low activity of GSTs containing Thr in position 210 is probably due to steric hindrance caused by the beta-methyl group of the side chain. The ratios of the different catalytic efficiencies were translated into differences in the Gibbs free energies of transition state stabilization. The effects of the mutations were qualitatively parallel for the alternative substrates, but vary significantly in magnitude. From the evolutionary perspective the data show that a point mutation can alternatively enhance or attenuate the activity with a particular substrate and illustrate the functional plasticity of GSTs.     

Crystallographic and Functional Characterization of the Fluorodifen-inducible Glutathione Transferase from Glycine max Reveals an Active Site Topography Suited for Diphenylether Herbicides and a Novel L-site

Glutathione transferases (GSTs) from the tau class (GSTU) are unique to plants and have important roles in stress tolerance and the detoxification of herbicides in crops and weeds. A fluorodifen-induced GST isoezyme (GmGSTU4-4) belonging to the tau class was purified from Glycine max by affinity chromatography. This isoenzyme was cloned and expressed in Escherichia coli, and its structural and catalytic properties were investigated. The structure of GmGSTU4-4 was determined at 1.75 Å resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). The enzyme adopts the canonical GST fold but with a number of functionally important differences. Compared with other plant GSTs, the three-dimensional structure of GmGSTU4-4 primarily shows structural differences in the hydrphobic substrate binding site, the linker segment and the C-terminal region. The X-ray structure identifies key amino acid residues in the hydrophobic binding site (H-site) and provides insights into the substrate specificity and catalytic mechanism of the enzyme. The isoenzyme was highly active in conjugating the diphenylether herbicide fluorodifen. A possible reaction pathway involving the conjugation of glutathione with fluorodifen is described based on site-directed mutagenesis and molecular modeling studies. A serine residue (Ser13) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione and enhance its nucleophilicity. Tyr107 and Arg111 present in the active site are important structural moieties that modulate the catalytic efficiency and specificity of the enzyme, and participate in k_cat regulation by affecting the rate-limiting step of the catalytic reaction. A hitherto undescribed ligand-binding site (L-site) located in a surface pocket of the enzyme was also found. This site is formed by conserved residues, suggesting it may have an important functional role in the transfer and delivery of bound ligands, presumably to specific protein receptors.     

Enzymatic and structural characterisation of amphinase, a novel cytotoxic ribonuclease from Rana pipiens oocytes

Besides Onconase (ONC) and its V11/N20/R103-variant, oocytes of the Northern Leopard frog (Rana pipiens) contain another homologue of ribonuclease A, which we named Amphinase (Amph). Four variants (Amph-1-4) were isolated and sequenced, each 114 amino acid residues in length and N-glycosylated at two positions. Sequence identities (a) among the variants and (b) versus ONC are 86.8-99.1% and 38.2-40.0%, respectively. When compared with other amphibian ribonucleases, a typical pattern of cysteine residues is evident but the N-terminal pyroglutamate residue is replaced by a six-residue extension. Amph variants have relatively weak ribonucleolytic activity that is insensitive to human ribonuclease inhibitor protein (RI). Values of k(cat)/K(M) with hypersensitive fluorogenic substrates are 10(4) and 10(2)-fold lower than the maximum values exhibited by ribonuclease A and ONC, respectively, and there is little cytosine/uracil or adenine/guanine discrimination at the B(1) or B(2) subsites, respectively. Amph variants have cytotoxic activity toward A-253 carcinoma cells that requires intact ribonucleolytic activity. The glycan component has little or no influence over single-stranded RNA cleavage, RI evasion or cytotoxicity. The crystal structures of natural and recombinant Amph-2 (determined at 1.8 and 1.9 A resolution, respectively) reveal that the N terminus is unlikely to play a catalytic role (but an unusual alpha2-beta1 loop may do so) and the B(2) subsite is rudimentary. At the active site, structural features that may contribute to the enzyme's low ribonucleolytic activity are the fixture of Lys14 in an obstructive position, the accompanying ejection of Lys42, and a lack of constraints on the conformations of Lys42 and His107.     

Engineering GST M2-2 for high activity with indene 1,2-oxide and indication of an H-site residue sustaining catalytic promiscuity

The substrate-binding H-site of human glutathione transferase (GST) M2-2 was subjected to iterative saturation mutagenesis in order to obtain an efficient enzyme with the novel epoxide substrate indene 1,2-oxide. Residues 10, 116, and 210 were targeted, and the activities with the alternative substrates, benzyl isothiocyanate and the prodrug azathioprine, undergoing divergent chemical reactions were monitored for comparison. In general, increased activities were found when the smaller residues Gly, Ser, and Ala replaced the original Thr210. The most active mutant T210G was further mutated at position 116, but no mutant showed enhanced catalytic activity. However, saturation mutagenesis of position 10 identified one double mutant T210G/I10C with 100-fold higher specific activity with indene 1,2-oxide than wild-type GST M2-2. This enhanced epoxide activity of 50 μmol min^− 1 mg^− 1 resulted primarily from an increased k_cat value (70 s^− 1). The specific activity is 24-fold higher than that of wild-type GST M1-1, which is otherwise the most proficient GST enzyme with epoxide substrates. A second double mutant T210G/I10W displayed 30-fold increased activity with azathioprine, 0.56 μmol min^− 1 mg^− 1. In both double mutants, the replacement of Ile10 led to narrowed acceptance of alternative substrates. Ile10 is evolutionarily conserved in related class Mu GSTs. Conservation usually indicates preservation of a particular function, and in the Mu class, it would appear that the conserved Ile10 is not necessary to maintain catalytic functions but to prevent loss of broad substrate acceptance. In summary, our data underscore the facile transition between alternative substrate selectivity profiles in GSTs by a few mutations.     

Structure of a Drosophila sigma class glutathione S-transferase reveals a novel active site topography suited for lipid peroxidation products

Insect glutathione-S-transferases (GSTs) are grouped in three classes, I, II and recently III; class I (Delta class) enzymes together with class III members are implicated in conferring resistance to insecticides. Class II (Sigma class) GSTs, however, are poorly characterized and their exact biological function remains elusive. Drosophila glutathione S-transferase-2 (GST-2) (DmGSTS1-1) is a class II enzyme previously found associated specifically with the insect indirect flight muscle. It was recently shown that GST-2 exhibits considerable conjugation activity for 4-hydroxynonenal (4-HNE), a lipid peroxidation product, raising the possibility that it has a major anti-oxidant role in the flight muscle. Here, we report the crystal structure of GST-2 at 1.75A resolution. The GST-2 dimer shows the canonical GST fold with glutathione (GSH) ordered in only one of the two binding sites. While the GSH-binding mode is similar to other GST structures, a distinct orientation of helix alpha6 creates a novel electrophilic substrate-binding site (H-site) topography, largely flat and without a prominent hydrophobic-binding pocket, which characterizes the H-sites of other GSTs. The H-site displays directionality in the distribution of charged/polar and hydrophobic residues creating a binding surface that explains the selectivity for amphipolar peroxidation products, with the polar-binding region formed by residues Y208, Y153 and R145 and the hydrophobic-binding region by residues V57, A59, Y211 and the C-terminal V249. A structure-based model of 4-HNE binding is presented. The model suggest that residues Y208, R145 and possibly Y153 may be key residues involved in catalysis.     

2,4-Dichlorophenoxyacetic Acid and Related Chlorinated Compounds Inhibit Two Auxin-Regulated Type-III Tobacco Glutathione S-Transferases

Two auxin-inducible glutathione S-transferase (GST, EC 2.5.1.18) isozymes from tobacco (Nicotiana tabacum, White Burley) were partially characterized. GST1-1 and GST2-1 are members of a recently identified new type of plant GST isozymes that we will here refer to as type III. Both enzymes were active, with 1-chloro-2,4-dinitrobenzene as a substrate, when expressed in bacteria as fusion proteins. The apparent Km for 1-chloro-2,4-dinitrobenzene was found to be 0.85 [plus or minus] 0.25 mM for GST1-1 and 0.20 [plus or minus] 0.15 mM for GST2-1. The apparent Km for glutathione was similar for both enzymes, 0.40 [plus or minus] 0.15 mM. The in vitro activity of both enzymes could be inhibited by the synthetic auxin 2,4-dichlorophenoxyacetic acid, with an apparent Ki of 80 [plus or minus] 40 [mu]M for GST1-1 and 200 [plus or minus] 100 [mu]M for GST2-1. The GST1-1 was also inhibited by structurally related substances, such as 2,4-dichlorobenzoic acid, with a roughly similar Ki. The nonchlorinated structures benzoic acid and phenoxyacetic acid did not inhibit. p-Chloroisobutyric acid, or clofibric acid, an auxin-transport inhibitor, was found to be an active inhibitor as well. The strongest inhibitor identified, however, was a phenylacetic acid derivative, ethacrynic acid, which showed an apparent Ki of 5 [plus or minus] 5 [mu]M for both enzymes. This substance is a known inducer as well as a substrate of specific mammalian GSTs. The results presented here indicate that the type III plant GSTs might be involved in the metabolism or transport of chlorinated substances that are structurally related to auxins. The possibility that auxins are endogenous ligands or substrates for GSTs is discussed.     

Comparative genomics identifies new alpha class genes within the avian glutathione S-transferase gene cluster

Glutathione S-transferases (GSTs: EC2.5.1.18) are a superfamily of multifunctional dimeric enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic chemicals. In most animals and in humans, GSTs are the principal enzymes responsible for detoxifying the mycotoxin aflatoxin B₁ (AFB₁) and GST dysfunction is a known risk factor for susceptibility towards AFB₁. Turkeys are one of the most susceptible animals known to AFB₁, which is a common contaminant of poultry feeds. The extreme susceptibility of turkeys is associated with hepatic GSTs unable to detoxify the highly reactive and electrophilic metabolite exo-AFB₁-8,9-epoxide (AFBO). In this study, comparative genomic approaches were used to amplify and identify the α-class tGST genes (tGSTA1.1, tGSTA1.2, tGSTA1.3, tGSTA2, tGSTA3 and tGSTA4) from turkey liver. The conserved GST domains and four α-class signature motifs in turkey GSTs (with the exception of tGSTA1.1 which lacked one motif) confirm the presence of hepatic α-class GSTs in the turkey. Four signature motifs and conserved residues found in α-class tGSTs are (1) xMExxxWLLAAAGVE, (2) YGKDxKERAxIDMYVxG, (3) PVxEKVLKxHGxxxL and (4) PxIKKFLXPGSxxKPxxx. A BAC clone containing the α-class GST gene cluster was isolated and sequenced. The turkey α-class GTS genes genetically map to chromosome MGA2 with synteny between turkey and human α-class GSTs and flanking genes. This study identifies the α-class tGST gene cluster and genetic markers (SNPs, single nucleotide polymorphisms) that can be used to further examine AFB₁ susceptibility and resistance in turkeys. Functional characterization of heterologously expressed proteins from these genes is currently underway.     

Directed evolution of sulfotransferases and paraoxonases by ancestral libraries

Large libraries of randomly mutated genes are applied in directed evolution experiments in order to obtain sufficient variability. These libraries, however, contain mostly inactive variants, and the very low frequency of improved variants can only be isolated by high-throughput screening. Small but efficient libraries comprise an attractive alternative. Here, we describe the application of ancestral libraries—libraries based on mutations predicted by phylogenetic analysis and ancestral inference. We designed and constructed such libraries using serum paraoxonases and cytosolic sulfotransferases (SULTs) as model enzymes. Both of these enzyme families exhibit a range of activities in drug metabolism and detoxification of xenobiotics. The ancestral serum paraoxonase and SULT libraries were screened by low-throughput means, including HPLC, using substrates and/or reactions with which all family members exhibit low activity. The libraries showed a remarkably high frequency of highly polymorphic and functionally diverse variants. Screening of as few as 300 variants enabled the isolation of mutants with up to 50-fold higher activity than the starting point enzyme. Structural and kinetic characterizations of an evolved SULT variant show how few ancestral mutations reshaped the active site and modulated the enzyme's specificity. Ancestral libraries therefore comprise a means of focusing diversity to positions and mutations that readily trigger changes in substrate and/or reaction specificity, thereby facilitating the isolation of new enzyme variants for a variety of different substrates and reactions by medium-throughput or even low-throughput screens.     

An ensemble of theta class glutathione transferases with novel catalytic properties generated by stochastic recombination of fragments of two mammalian enzymes

The correlation between sequence diversity and enzymatic function was studied in a library of Theta class glutathione transferases (GSTs) obtained by stochastic recombination of fragments of cDNA encoding human GST T1-1 and rat GST T2-2. In all, 94 randomly picked clones were characterized with respect to sequence, expression level, and catalytic activity in the conjugation reactions between glutathione and six alternative electrophilic substrates. Out of these six different compounds, dichloromethane is a selective substrate for human GST T1-1, whereas 1-menaphthyl sulfate and 1-chloro-2,4-dinitrobenzene are substrates for rat GST T2-2. The other three substances serve as substrates for both enzymes. Through this broad characterization, we have identified enzyme variants that have acquired novel activity profiles that differ substantially from those of the original GSTs. In addition, the expression levels of many clones were improved in comparison to the parental enzyme. A library of mutants can thus display a distribution of properties from which highly divergent evolutionary pathways may emerge, resembling natural evolutionary processes. From the GST library, a clone was identified that, by the point mutation N49D in the rat GST T2-2 sequence, has a 1700% increased activity with 1-menaphthyl sulfate and a 60% decreased activity with 4-nitrophenethyl bromide. Through the N49D mutation, the ratio of these activities has thus been altered 40-fold. An extensive characterization of a population of stochastically mutated enzymes can accordingly be used to find variants with novel substrate-activity profiles and altered catalytic properties. Recursive recombination of selected sequences displaying optimized properties is a strategy for the engineering of proteins for medical and biochemical applications. Such sequential design is combinatorial protein chemistry based on remodeling of existing structural scaffolds and has similarities to evolutionary processes in nature.     

Beta sheet 2–alpha helix C loop of cytochrome P450 reductase serves as a docking site for redox partners

As a promiscuous redox partner, the biological role of cytochrome P450 reductase (CPR) depends significantly on protein–protein interactions. We tested a hypothesized CPR docking site by mutating D113, E115, and E116 to alanine and assaying activity toward various electron acceptors as a function of ionic strength. Steady-state cytochrome c studies demonstrated the mutations improved catalytic efficiency and decreased the impact of ionic strength on catalytic parameters when compared to wild type. Based on activity toward 7-ethoxy-4-trifluoro-methylcoumarin, CYP2B1 and CPR favored formation of an active CYP2B1•CPR complex and inactive (CYP2B1)₂•CPR complex until higher ionic strength whereby only the binary complex was observed. The mutations increased dissociation constants only for the binary complex and suppressed the ionic strength effect. Studies with a non-binding substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) suggest changes in activity toward cytochrome c and CYP2B1 reflect alterations in the route of electron transfer caused by the mutations. Electrostatic modeling of catalytic and binding parameters confirmed the importance of D113 and especially the double mutant E115 and E116 as mediators in forming charge–charge interactions between CPR and complex partners.