Random Chimeragenesis Research Articles

Developments in Directed Evolution for Improving Enzyme Functions

The engineering of enzymes with altered activity, specificity, and stability, using directed evolution techniques that mimic evolution on a laboratory timescale, is now well established. In vitro recombination techniques such as DNA shuffling, staggered extension process (StEP), random chimeragenesis on transient templates (RACHITT), iterative truncation for the creation of hybrid enzymes (ITCHY), recombined extension on truncated templates (RETT), and so on have been developed to mimic and accelerate nature's recombination strategy. This review discusses gradual advances in the techniques and strategies used for the directed evolution of biocatalytic enzymes aimed at improving the quality and potential of enzyme libraries, their advantages, and disadvantages.

Recognition of Phospholipids in Streptomyces Phospholipase D

To investigate the contribution of amino acid residues to the enzyme reaction of Streptomyces phospholipase D (PLD), we constructed a chimeric gene library between two highly homologous plds, which indicated different activity in transphosphatidylation, using RIBS (repeat-length independent and broad spectrum) in vivo DNA shuffling. By comparing the activities of chimeras, six candidate residues related to transphosphatidylation activity were shown. Based on the above result, we constructed several mutants to identify the key residues involved in the recognition of phospholipids. By kinetic analysis, we identified that Gly188 and Asp191 of PLD from Streptomyces septatus TH-2, which are not present in the highly conserved catalytic HXKXXXXD (HKD) motifs, are key amino acid residues related to the transphosphatidylation activity. To investigate the role of two residues in the recognition of phospholipids, the effects of these residues on binding to substrates were analyzed by surface plasmon spectroscopy. The result suggests that Gly188 and Asp191 are involved in the recognition of phospholipids in correlation with the N-terminal HKD motif. Furthermore, this study also provides experimental evidence that the N-terminal HKD motif contains the catalytic nucleophile, which attacks the phosphatidyl group of the substrate.

Repeat-Length-Independent Broad-Spectrum Shuffling, a Novel Method of Generating a Random Chimera Library In Vivo

We describe a novel method of random chimeragenesis based on highly frequent deletion formation in the Escherichia coli ssb-3 strain and a deletion-directed chimera selection system that uses the rpsL(+) gene as a reporter. It enables the selection of chimeras without target gene expression and can therefore be applied to cytotoxic targets. When this system was applied to phospholipase D genes from Streptomyces septatus TH-2 and Streptomyces halstedii subsp. scabies K6 (examples of cytotoxic targets), chimeragenesis occurred between short identical sequences at the corresponding position of the parental genes with large variations. Chimeragenesis was >1,000 times more frequent in the ssb-3 background than in the ssb(+) background. We called this system repeat-length-independent broad-spectrum shuffling. It enables the convenient chimeragenesis and functional study of chimeric proteins. In fact, we found two amino acid residues related to the thermostability of phospholipase D (Phe426 and Thr433) by comparing thermostability among the chimeric enzymes obtained.

Identification of amino acids in rat pregnane X receptor that determine species-specific activation.

The pregnane X receptor (PXR) is a nuclear receptor significantly involved in the transcriptional regulation of drug-metabolizing enzymes and transporters. Interestingly, certain PXR ligands such as rifampin have been shown to readily induce human and rabbit but not rodent members of the cytochrome P450 3A. Because drugs of divergent chemical structures seem to be similarly affected, we hypothesized that specific amino acid residue(s) or domains in rat PXR affect receptor activation by certain human PXR ligands. To identify such a domain(s), an array of human-rat and rat-human chimeric PXR cDNAs in a tandem head-to-tail configuration were created using a random chimeragenesis method. Pharmacological characterization of these chimeras revealed a discreet segment within the ligand-binding domain of rat and human PXR to be essential for the rifampin effect. Within this region, the corresponding residues Leu308 and Phe305 of human and rat PXR, respectively, were found to be important for rifampin activation. Homology modeling derived from the recently determined crystal structure of human PXR indicates that these amino acids are located within or neighboring the flexible loop that forms part of the pore to the ligand-binding cavity. Rifampin, paclitaxel, and hyperforin sensitivity was conferred to rat PXR when Phe305 was converted to leucine, whereas attenuation of sensitivity was observed when Leu308 of human PXR was replaced with phenylalanine. Accordingly, our data provide compelling new insight into the importance of the amino acids comprising the pore to the ligand-binding cavity as a critical modulator of PXR response.

Chemostat approach for the directed evolution of biodesulfurization gain-of-function mutants.

Chemostat enrichment is a classical microbiological method that is well suited for use in directed-evolution strategies. We used a two-phase sulfur-limited chemostat to select for gain-of-function mutants with mutations in the biodesulfurization (Dsz) system of Rhodococcus erythropolis IGTS8, enriching for growth in the presence of organosulfur compounds that could not support growth of the wild-type strain. Mutations arose that allowed growth with octyl sulfide and 5-methylbenzothiophene as sole sulfur sources. An isolate from the evolved chemostat population was genetically characterized and found to contain mutations in two genes, dszA and dszC. A transversion (G to T) in dszC codon 261 resulted in a V261F mutation that was determined to be responsible for the 5-methylbenzothiophene gain-of-function phenotype. By using a modified RACHITT (random chimeragenesis on transient templates) method, mutant DszC proteins containing all possible amino acids at that position were generated, and this mutant set was assayed for the ability to metabolize 5-methylbenzothiophene, alkyl thiophenes, and dibenzothiophene. No mutant with further improvements in these catalytic activities was identified, but several clones lost all activity, confirming the importance of codon 261 for enzyme activity.

27] Random chimeragenesis by heteroduplex recombination

Publisher Summary Genetic variations existing in nature or created in the laboratory can be recombined to generate libraries of molecules containing novel combinations of sequence information from any or all of the parent sequences. By combining beneficial mutations and removing deleterious ones, recombination may help to accelerate the evolution of single molecules toward a specified function. Novel chimeric sequences generated by the recombination of homologous, naturally occurring genes also provide an extremely rich source of genetic diversity for directed evolution. This chapter describes a simple method for creating libraries of chimeric deoxyribonucleic acid (DNA) sequences derived from homologous parental sequences that uses in vivo repair of heteroduplexes for recombination. This heteroduplex recombination approach relies on the mismatch repair system of the host cells to repair regions of nonidentity in the heteroduplex and creates a library of new sequences composed of elements from each parent. This method combines the advantages of in vitro and in vivo recombination methods and avoids some of their drawbacks. Heteroduplex recombination does not require polymerase chain reaction (PCR) amplification and should be useful for the recombination of large DNA sequences.

26] Methods for in vitro DNA recombination and random chimeragenesis

Publisher Summary In vitro polymerase chain reaction (PCR)-based methods for recombining homologous deoxyribonucleic acid (DNA) sequences are capable of creating highly mosaic chimeric sequences. Several different methods have been reported for in vitro recombination or DNA shuffling: for example, the original Stemmer method of DNase I fragmentation and reassembly, the staggered extension process (STEP), and random priming recombination. Slight variations in the shuffling protocols can affect the outcome of the experiment. Different gene sequences recombine most efficiently under different conditions. This chapter provides protocols that are designed to give a high likelihood of success. The protocols are known to work for recombining sequences of ∼>85% identity.

Identification of Amino Acid Determinants of the Positional Specificity of Mouse 8S-Lipoxygenase and Human 15S-Lipoxygenase-2

Phorbol ester-inducible mouse 8S-lipoxygenase (8-LOX) and its human homologue, 15S-lipoxygenase-2 (15-LOX-2), share 78% identity in amino acid sequences, yet there is no overlap in their positional specificities. In this study, we investigated the determinants of positional specificity using a random chimeragenesis approach in combination with site-directed mutagenesis. Exchange of the C-terminal one-third of the 8-LOX with the corresponding portion of 15-LOX-2 yielded a chimeric enzyme with exclusively 15S-lipoxygenase activity. The critical region was narrowed down to a cluster of five amino acids by expression of multiple cDNAs obtained by in situ chimeragenesis in Escherichia coli. Finally, a pair of amino acids, Tyr(603) and His(604), was identified as the positional determinant by site-directed mutagenesis. Mutation of both of these amino acids to the corresponding amino acids in 15-LOX-2 (Asp and Val, respectively) converted the positional specificity from 8S to 90% 15S without yielding any other by-products. Mutation of the corresponding residues in 15-LOX-2 to the 8-LOX sequence changed specificity to 50% oxygenation at C-8 for one amino acid substitution and 70% at C-8 for the double mutant. Based on the crystal structure of the reticulocyte 15-LOX, these two amino acids lie opposite the open coordination position of the catalytic iron in a likely site for substrate binding. The change from 8 to 15 specificity entails a switch in the head to tail binding of substrate. Enzymes that react with substrate "head first" (5-LOX and 8-LOX) have a bulky aromatic amino acid and a histidine in these positions, whereas lipoxygenases that accept substrates "tail first" (12-LOX and 15-LOX) have an aliphatic residue with a glutamine or aspartate. Thus, this positional determinant of the 8-LOX and 15-LOX-2 may have significance for other lipoxygenases.

The Use of Random Chimeragenesis to Study Structure/Function Properties of Rat and Human P450c17

The microsomal 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17) catalyzes the 17α-hydroxylase reaction required to produce cortisol, the major glucocorticoid in many species and the 17,20-lyase activity required for the production of androgens in all species. Utilizing the technique of random chimeragenesis we have attempted to map regions of primary sequence that contribute to the species-specific biochemical differences between rat and human P450c17. We have previously reported significant differences between rat and human P450c17 in their activities, stability and substrate-dependent coupling efficiencies even though they share 68% amino acid identity. Identification of the regions of primary sequence that contribute to each of these properties would be helpful in understanding the structure/function relationships in this enzyme. A single plasmid containing the cDNAs encoding both enzymes in a tandem orientation was constructed. This plasmid was linearized at unique restriction sites and used to transform Escherichia coli. A three-step screening protocol identified five chimeras with a uniform distribution of 5′ rat and 3′ human sequence. All chimeric proteins yield the characteristic reduced-CO difference spectra, indicating proper folding. The chimeras exhibit a range of stability and activities that are not consistent with the degree of parental primary sequence. A chimera containing 301 N-terminal rat P450c17 amino acids and lacking the rat P450c17 phenylalanine 343, had the highest lyase activity. Generation of these functional rat/human chimeras suggests that the tertiary structures of rat and human P450c17 are sufficiently conserved to allow proper folding of chimeric enzymes. However, the properties of these chimeras did not permit identification of a region of primary sequence that contributes to a species-specific property of rat and human P450c17. Stability of these chimeras and insight into the presence of secondary structural elements is discussed.

CCR5 HIV-1 Coreceptor Activity: ROLE OF COOPERATIVITY BETWEEN RESIDUES IN N-TERMINAL EXTRACELLULAR AND INTRACELLULAR DOMAINS

Human (H-) CCR5 is the primary coreceptor for ENV-mediated fusion by R5 strains of human immunodeficiency virus type 1, whereas mouse (M-) CCR5 lacks this function. An array of 23 H/M-CCR5 hybrids containing increasing amounts of H-CCR5 extending from the N terminus generated by random chimeragenesis had a biphasic pattern of coreceptor activity with JRFL and 89.6, revealing active regions in the N-terminal extracellular domain (N-ED) and at the junction of cytoplasmic loop 3. The M-CCR5 mutant in which divergent residues were replaced with the corresponding H-CCR5 N-ED sequence (NyYTsE) gained coreceptor function in fusion but not infection experiments. A M-CCR5 double mutant with substitution of human sequences for divergent residues from the N-ED and cytoplasmic loop 3 had augmented coreceptor activity in fusion assays and gain of function in infection experiments. The SIV-251 ENV utilized H- and M-CCR5 and variants. Flow cytometric analysis of M-CCR5 mutants and bifunctional receptors composed of CD4 domains fused to M-CCR5 mutants excluded the possibility that differences in coreceptor activity resulted from variations in cell surface expression. These results demonstrate that the coreceptor activity of the H-CCR5 N-ED is modulated by intracellular residues, illustrating the complexity of CCR5 requirements for interaction with ENV.

Deletion of Ala144-Lys145 in Thermus thermophilus inorganic pyrophosphatase suppresses thermal aggregation.

The regions contributing to the thermostability of inorganic pyrophosphatase (PPase, EC 3.6.1.1) from Thermus thermophilus (Tth) were deduced in our previous study by random chimeragenesis, one of them being estimated to be Ala144-Lys145 [Satoh, T., Takahashi, Y., Oshida, N., Shimizu, A., Shinoda, H., Watanabe, M., and Samejima, T. (1999) Biochemistry 38, 1531-1536]. Therefore, we investigated the contributions of these two residues in Tth by preparing a deletion mutant (del.144-145 mutant) of Tth PPase. We examined its thermostability in terms of the CD and fluorescence spectra, and the thermal change in the enzymatic activity. The thermostability of the enzymatic activity of the del.144-145 mutant was similar to that of the wild type Tth PPase, whereas this mutant was more stable against heating. Furthermore, we compared the thermal aggregation of the wild type with that of the del.144-145 mutant. We found that the thermal aggregation of the mutant was reduced relative to that of the wild type. Moreover, the molecular weight of the mutant after heating at 90 degrees C was higher than that of the unheated one, whereas the wild type aggregated under the same conditions. Therefore, we can conclude that although the Ala144-Lys145 residues in Tth PPase may partly cause thermal aggregation, the deletion of these residues may stabilize the Tth PPase molecule structurally against heating and suppress thermal aggregation.

A chimeric inorganic pyrophosphatase derived from Escherichia coli and Thermus thermophilus has an increased thermostability.

Factors contributing to the thermostability of inorganic pyrophosphatase (PPase) were investigated by examining chimeric PPases from Escherichia coli and Thermus thermophilus (Tth). Two chimeric PPase genes, T1-135E (residues 1-135 from the N terminus are comprised of Tth PPase and residues 136-173 are derived from the C terminus of E. coli PPase) and T1-149E [residues 1-149 from the N terminus are from Tth PPase and the rest (150-175) are from E. coli PPase], were constructed by random chimeragenesis. After the genes were overexpressed in the E. coli BL21(DE3) strain and the expression products were purified, we compared the characteristics of these chimeric PPases with those of the parental PPases. We found that the two chimeras had higher activity than either parent PPase at the optimum temperature. We also examined thermal stability in terms of CD spectra, fluorescence spectra, and thermal changes in enzyme activity. The results revealed that the thermal stability of T1-149E is similar to that of Tth PPase, but T1-135E is much more stable. This suggests that the four residues that are different between T1-135E and T1-149E may be critical for thermostability between the two chimeras. By comparing the three-dimensional structures of Tth and E. coli PPases, we deduced that the following two factors may contribute to differences in thermostability. (1) Two residues (Thr138 and Ala141 in the Tth PPase and His140 and Asp143 in the E. coli PPase) in the vicinity of the trimer-trimer interface were different. (2) The Ala144-Lys145 loop in the Tth PPase was deleted in the E. coli PPase and also in the T1-135E chimera. Therefore, we conclude that T1-135E was thermostabilized by these two factors, and also, the Tth PPase moiety may contribute to the structural integrity of the chimeric enzymes.

Identification of amino acid residues associated with modulation of flavin-containing monooxygenase (FMO) activity by imipramine: structure/function studies with FMO1 from pig and rabbit.

The activity of the flavin-containing monooxygenase (FMO) can be modulated by a number of nitrogen-containing compounds in a manner that is both isoform and modulator-dependent. We now show that the direction (activation or inhibition) and extent of modulation can also be dependent on substrate concentration. Imipramine activates methimazole metabolism catalyzed by rabbit FMO1 or FMO2 at methimazole concentrations greater than 50 or 100 microM, respectively, and inhibits at lower methimazole concentrations. The extent of the activation increases as the substrate concentration increases, and the extent of inhibition increases as the substrate concentration decreases. With either inhibition or activation, the magnitude of the effect shows a similar, direct dependency on imipramine concentration. In contrast, imipramine inhibits the metabolism of methimazole catalyzed by pig FMO1 at all substrate concentrations. The structural basis for this unique ortholog difference between the responses of rabbit and pig FMO1 to imipramine was studied by random chimeragenesis and site-directed mutagenesis. Results with chimeras indicated that modulation of FMO1 activity by imipramine is controlled to a great extent by two areas of the FMO primary structure (residues 381-432 and 433-465). Four amino acids in these regions (positions 381, 400, 420 and 433) and one additional residue (position 186) were identified by site-directed mutagenesis as primary determinants of the imipramine response. When the residues at these positions in rabbit FMO1 are exchanged for the corresponding residues of pig FMO1, a mutant with the functional properties of pig FMO1 is produced. Our results suggest that the response of FMO1 to imipramine involves a distribution between two sites that is regulated by structural features that do not alter the overall binding. The inhibition observed, although it appears to be competitive, likely does not involve competition for a binding site since alteration of imipramine metabolism has no effect on the parameters of methimazole metabolism.

Studies of Distant Members of the P450 Superfamily (P450scc and P450c27) by Random Chimeragenesis

“Random chimeragenesis” (Kim, J.-Y., and Devreotes, P. N. (1994)J. Biol. Chem.269, 28724–28731; Levin, L. R., and Reed, R. R. (1995)J. Biol. Chem.270, 7573–7579) has been used to generate chimeras between two distant P450s involved in catabolism of cholesterol in mammals, P450scc (product of CYP11A1 gene) and P450c27 (product of CYP27 gene). Both are mitochondrial P450s which hydroxylate the side chain of cholesterol. Even though these P450s are only about 25% identical, we wondered whether their similar substrate specificity might permit mapping of the active sites by this technique. Four chimeric DNAs encoding three different proteins have been obtained testifying that short stretches of nucleotide identity (six nucleotides preceding the crossover point) are sufficient for homologous recombination inEscherichia coli,the basis of the random chimeragenesis technique. The N-terminal part of the chimeras was formed by P450scc and the C-terminus was from P450c27. The chimeric P450s have been expressed inE. coliand partially purified. Though they displayed a peak at 453 nm in their CO-difference spectra, indicating that the proteins are properly folded and hemin is correctly incorporated, none of the chimeras had detectable catalytic activity with either cholesterol (substrate for P450scc) or 5β-cholestane-3α,7α,12α-triol (substrate for P450c27). To investigate whether the chimeras bind substrate, titrations with 22R-hydroxycholesterol have been carried out. Addition of 22R-hydroxycholesterol to recombinant P450scc which is in the low spin form leads to conversion to the high spin form. Titration of chimeras with 22R-hydroxycholesterol did not result in their conversion to the high spin form. However, a shift to 426–430 nm was observed in the difference spectrum of two of the chimeras with a minimum around 406 nm. Thus, addition of 22R-hydroxycholesterol to these chimeras results in binding of the steroid in the enzyme active site and conversion of one low spin form to a different low spin form. Possible explanations for the absence of enzymatic activity and the potential advantages of the random chimeragenesis technique to generate chimeras between different P450s are discussed.

Genetically engineered bacterial cells and applications

Heterologous expression systems have proven to be very important in P450 research, permitting investigation of many P450s identified only by recombinant (cloning) technologies. They also make it possible to study human P450s since tissue sources for naturally occurring enzymes are difficult to obtain. A variety of different heterologous systems have been applied to the study of P450s and each one has its own unique advantages. For the study of biophysical properties and structure/function relationships, E. coli has proven to be a particularly good system. Both microsomal and mitochondrial P450s can be expressed to high levels in E. coli and subsequently purified to homogeneity for detailed analysis. Techniques of both site directed mutagenesis and random chimeragenesis are very facile in bacteria providing excellent opportunity to analyze specific aspects of structure/function relationships of P450s. Furthermore microsomal P450s are active in intact E. coli, activities being supported by flavodoxin and flavodoxin reductase, providing the opportunity to develop bioreactors expressing designer P450s. The salient features of P450 expression in bacteria are summarized herein.

Random chimeragenesis of G-protein-coupled receptors. Mapping the affinity of the cAMP chemoattractant receptors in Dictyostelium.

G-protein-coupled receptors mediate a wide variety of responses to extracellular stimuli in eucaryotic cells. Binding of the ligand to these receptors is thought to involve contacts within a pocket from the sequences of these genes. A family of four surface cAMP receptors that mediate responses to secreted cAMP coordinates the developmental program of Dictyostelium. A large difference in affinity for cAMP exists between cAR1 (25 and 230 nM) and cAR2 (> 5 microns). To understand the basis for this affinity difference, we generated an extensive series of cAR1/cAR2 and cAR2/cAR1 chimeras using a technique designated "random chimeragenesis." When a linearized plasmid was transformed into Escherichia coli, tandemly positioned cAR1 and cAR2 genes crossed over at homologous regions. The cAMP binding properties and EC50 values for agonist-induced phosphorylation of each of the chimeras were characterized in order to map the domains that determine the affinity. These studies implicated a domain in the second extracellular loop in which only 5 residues differ between the two receptors as the major determinant of affinity. A secondary domain including residues 110-147 (11 residue differences) was identified as a minor determinant of affinity.