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PubMed2005

Directed evolution of a ring-cleaving dioxygenase for polychlorinated biphenyl degradation.

DoxG, an extradiol dioxygenase involved in the aerobic catabolism of naphthalene, possesses a weak ability to cleave 3,4-dihydroxybiphenyls (3,4-DHB), critical polychlorinated biphenyl metabolites. A directed evolution strategy combining error-prone PCR, saturation mutagenesis, and DNA shuffling was used to improve the polychlorinated biphenyl-degrading potential of DoxG. Screening was facilitated through analysis of filtered, digital imaging of plated colonies. A simple scheme, which is readily adaptable to other activities, enabled the screening of >10(5) colonies/h. The best variant, designated DoxGSMA2, cleaved 3,4-DHB with an apparent specificity constant of 2.0 +/- 0.3 x 10(6) m(-1) s(-1), which is 770 times that of wild-type (WT) DoxG. The specificities of DoxGSMA2 for 1,2-DHN and 2,3-DHB were increased by 6.7-fold and reduced by 2-fold, respectively, compared with the WT enzyme. DoxGSMA2 contained three substituted residues with respect to the WT enzyme: L190M, S191W, and L242S. Structural data indicate that the side chains of residues 190 and 242 occur on opposite walls of the substrate binding pocket and may interact directly with the distal ring of 3,4-DHB or influence contacts between this substrate and other residues. Thus, the introduction of two bulkier residues on one side of the substrate binding pocket and a smaller residue on the other may reshape the binding pocket and alter the catalytically relevant interactions of 3,4-DHB with the enzyme and dioxygen. Kinetic analyses reveal that the substitutions are anti-cooperative.

Base Sequence↗

PubMed2003

In vitro evolution of the binding specificity of neocarzinostatin, an enediyne-binding chromoprotein.

Neocarzinostatin is the most studied member of the enediyne-chromoprotein family, and is clinically used as an antitumoral agent. Neocarzinostatin could be a promising drug delivery vehicle if new binding specificities could be conferred to its protein scaffold. We used in vitro evolution methods to demonstrate that this approach is feasible. We created large libraries containing between 1.7 x 10(8) and 1.4 x 10(9) independent clones, where up to 13 side chains pointing toward the binding crevice were randomly substituted. We then used phage display to select variants that bind to a model ligand (testosterone) which is unrelated to the natural ligand of neocarzinostatin. Several different binders were selected from each library. The corresponding proteins were expressed in Escherichia coli and their affinities and specificities were characterized in detail. K(D) values of about 20 nM were obtained for streptavidin-bound testosterone. The K(D) of selected proteins for free soluble testosterone are between 7 and 55 microM and therefore higher than the K(D) for streptavidin-bound testosterone. The spacer and streptavidin used during selection contributed to the high affinity of the selected binders for the target. Binding studies of 15 different steroids related to testosterone allowed us to determine that C3, 4, 5, 6, and 7 on cycles A and B and the conjugated 3 oxo group of the steroid molecule were essential for molecular recognition. Other testosterone analogues substituted on C1, 2, 9, 11, 15, and 17 were not discriminated from testosterone. These results demonstrate that the binding specificity of this protein family can be extended to compounds that are completely unrelated to the natural enediyne chromophore family. This type of highly expressed, stable proteins with tailored binding properties have a wide potential range of applications.

Amino Acid Sequence↗

PubMed2004

Modulating D-amino acid oxidase substrate specificity: production of an enzyme for analytical determination of all D-amino acids by directed evolution.

Recent research on the flavoenzyme D-amino acid oxidase from Rhodotorula gracilis (RgDAAO) has revealed new, intriguing properties of this catalyst and offers novel biotechnological applications. Among them, the reaction of RgDAAO has been exploited in the analytical determination of the D-amino acid content in biological samples. However, because the enzyme does not oxidize acidic D-amino acids, it cannot be used to detect the total amount of D-amino acids. We now present the results obtained using a random mutagenesis approach to produce RgDAAO mutants with a broader substrate specificity. The libraries of RgDAAO mutants were generated by error-prone PCR, expressed in BL21(DE3)pLysS Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. Five random mutants that have a 'modified' substrate specificity, more useful for the analytical determination of the entire content of D-amino acids than wild-type RgDAAO, have been isolated. With the only exception of Y223 and G199, none of the effective amino acid substitutions lie in segments predicted to interact directly with the bound substrate. The substitutions appear to cluster on the protein surface: it would not have been possible to predict that these substitutions would enhance DAAO activity. We can only conclude that these substitutions synergistically generate small structural changes that affect the dynamics and/or stability of the protein in a way that enhances substrate binding or subsequently catalytic turnover.

Alanine↗

PubMed2005

Activity-based in vitro selection of T4 DNA ligase.

Recent in vitro methodologies for selection and directed evolution of proteins have concentrated not only on proteins with affinity such as single-chain antibody but also on enzymes. We developed a display technology for selection of T4 DNA ligase on ribosome because an in vitro selection method for DNA ligase had never been developed. The 3' end of mRNA encoding the gene of active or inactive T4 DNA ligase-spacer peptide fusion protein was hybridized to dsDNA fragments with cohesive ends, the substrate of T4 DNA ligase. After in vitro translation of the mRNA-dsDNA complex in a rabbit reticulocyte system, a mRNA-dsDNA-ribosome-ligase complex was produced. T4 DNA ligase enzyme displayed on a ribosome, through addition of a spacer peptide, is able to react with dsDNA in the complex. The complex expressing active ligase was biotinylated by ligation with another biotinylated dsDNA probe and selected with streptavidin-coated magnetic beads. We effectively selected active T4 DNA ligase from a small amount of protein. The gene of the active T4 DNA ligase was enriched 40 times from a mixture of active and inactive genes using this selection strategy. This ribosomal display strategy may have high potential to be useful for selection of other enzymes associated with DNA.

Catalysis↗

PubMed2006

Four-base codon mediated mRNA display to construct peptide libraries that contain multiple nonnatural amino acids.

In vitro selection and directed evolution of peptides from mRNA display are powerful strategies to find novel peptide ligands that bind to target biomolecules. In this study, we expanded the mRNA display method to include multiple nonnatural amino acids by introducing three different four-base codons at a randomly selected single position on the mRNA. Another nonnatural amino acid may be introduced by suppressing an amber codon that may appear from a (NNK)(n) nucleotide sequence on the mRNA. The mRNA display was expressed in an Escherichia coli in vitro translation system in the presence of three types of tRNAs carrying different four-base anticodons and a tRNA carrying an amber anticodon, the tRNAs being chemically aminoacylated with different nonnatural amino acids. The complexity of the starting mRNA-displayed peptide library was estimated to be 1.1 x 10(12) molecules. The effectiveness of the four-base codon mediated mRNA display method was demonstrated in the selection of biocytin-containing peptides on streptavidin-coated beads. Moreover, a novel streptavidin-binding nonnatural peptide containing benzoylphenylalanine was obtained from the nonnatural peptide library. The nonnatural peptide library from the four-base codon mediated mRNA display provides much wider functional and structural diversity than conventional peptide libraries that are constituted from 20 naturally occurring amino acids.

Amino Acids↗

PubMed2005

Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution.

DNA shuffling is widely used for optimizing complex properties contained within DNA and proteins. Demonstrated here is the amplification of a gene library by PCR using uridine triphosphate (dUTP) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides. The incorporated uracil bases were excised using uracil-DNA-glycosylase and the DNA backbone subsequently cleaved with piperidine. These end-point reactions required no adjustments. Polyacrylamide urea gels demonstrated adjustable fragmentation size over a wide range. The oligonucleotide pool was reassembled by internal primer extension to full length with a proofreading polymerase to improve yield over Taq. We present a computer program that accurately predicts the fragmentation pattern and yields all possible fragment sequences with their respective likelihood of occurrence, taking the guesswork out of the fragmentation. The technique has been demonstrated by shuffling chloramphenicol acetyltransferase gene libraries. A 33% dUTP PCR resulted in shuffled clones with an average parental fragment size of 86 bases even without employment of a fragment size separation, and revealed a low mutation rate (0.1%). NExT DNA fragmentation is rational, easily executed and reproducible, making it superior to other techniques. Additionally, NExT could feasibly be applied to several other nucleotide analogs.

Chloramphenicol O-Acetyltransferase↗

PubMed2006

High efficiency and throughput system in directed evolution in vitro of reporter gene.

In vitro directed evolution, especially with DNA shuffling, is a powerful means in biological studies of protein structure and function, and consequently for industrial applications. Escherichia coli beta-glucuronidase (gusA) gene, a versatile and efficient reporter gene, was the model for studying in vitro directed evolution because of its stability, easy analysis of the enzyme properties and conveniently visible phenotype. We developed a high efficiency, throughput system for in vitro directed evolution using gusA reporter gene as the model. The system consisted mainly of three aspects: a prokaryotic expression vector pYPX251, an easy method for obtaining the mutated gene from DNA shuffling and a suitable selected strategy. The vector pYPX251 carried the moderately strong aacC1 gene promoter and T1T2 transcription terminator that allowed expression in E. coli. Over 10,000 individuals could be selected individually in a 9 cm Petri dish after colonies were absorbed on a nitrocellulose filter. A library, which contained 100,000 individuals was screened by incubating ten filter papers with X-Glu. The polymerase chain reaction products of the gusA gene, the fragments of 50-100 bp, with high mutation rates were purified using a dialysis bag from 10% PAGE after electrophoresis. The possibility of obtaining desirable mutations was increased dramatically as the size of the library expanded. A GUS variant, named GUS-TR, was obtained through this system, which is significantly more resistant to high temperature than the wild type enzyme. GUS-TR maintained its high activity even when the nitrocellulose filter containing the variant colony was heated at 100 degrees C for 30 min.

Biotechnology↗

PubMed2003

Enhanced synthesis of poly(3-hydroxybutyrate) in recombinant Escherichia coli by means of error-prone PCR mutagenesis, saturation mutagenesis, and in vitro recombination of the type II polyhydroxyalkanoate synthase gene.

Type II synthase (PhaC1(Ps)) for polyhydroxyalkanoate (PHA) from Pseudomonas sp. 61-3 was subjected to an in vitro evolution system including PCR-mediated mutagenesis in order to improve the function of PhaC1(Ps) in terms of its ability to produce poly(3-hydroxybutyrate) [P(3HB)] in recombinant Escherichia coli. Based on our established in vivo assay system, two positions (Ser325 and Gln481) where mutations provided remarkable increases in P(3HB) synthesis were identified. Saturation mutagenesis at these positions was carried out to explore whether there might be more beneficial sequences for P(3HB) synthesis than those identified in the point mutation library. As a result, five single mutants [S325C (T) and Q481M (K, R)] gave rise to highly enhanced P(3HB) synthesis. Drastically enhanced P(3HB) synthesis (up to 340- to 400-fold the amount of that of the wild type) was further achieved by generation of all five variants of the double mutants combining the codons for residues 325/481. It is feasible that the replacement of Ser (specific for type II synthase) by Thr (specific for type I synthase) at position 325 resulted in acquiring greater P(3HB) synthesis ability as exhibited by type I synthases. The other hot spot, 481, that positively contributes to enhanced P(3HB) synthesis is located adjacent to a His479, a residue that forms a putative catalytic diad that can be inferred by sequence alignment.

Acyltransferases↗

PubMed1998

Directed evolution of new enzymes and pathways for environmental biocatalysis.

Biocatalysis is important in both natural and engineered environments. The major global reactions in the biospheric cycling of carbon, nitrogen, and other elements are catalyzed by microorganisms. The global carbon cycle includes millions of organic compounds that are made by plants, microorganisms, and organic chemists. Most of those compounds are transformed by microbial enzymes. Degradative metabolism is known as catabolism and yields principally carbon dioxide, methane, or biomass. Microbial catabolic enzymes are a great resource for biotechnology. They are the building blocks for engineering novel metabolic pathways and evolving improved enzymes in the laboratory. Two multicomponent bacterial oxygeneases, cytochrome P450cam and toluene dioxygenase, catalyze the dechlorination of polyhalogenated C2 compounds. Seven genes encoding those functional enzyme complexes were coexpressed in a Pseudomonas and shown to metabolize pentachloreothane to nonhalogenated organic acids that were metabolized further to carbon dioxide. In another example, the enzyme catalyzing the dechlorination of the herbicide atrazine was subjected to iterative DNA shuffling to produce mutations. By using a plate screening assay, mutated atrazine chlorohydrolase that catalyzed a more rapid dechlorination of atrazine was obtained. The mutant genes were sequences and found to encode up to 11 amino acid changes. Atrazine chlorohydrolase is currently being used in a model municipal water treatment system to test the feasibility of using enzymes for atrazine decontamination. These data suggest that the natural diversity of bacterial catabolic enzymes provides the starting point for improved biocatalytic systems that meet the needs of commercial applications.

Biodegradation, Environmental↗

PubMed2001

Fending off decay: a combinatorial approach in intact cells for identifying mRNA stability elements.

The strategy of systematic evolution, whereby nucleic acid sequences or conformers can be selected and amplified from a randomized population, has been exploited by many research groups for numerous purposes. It is, however, a technique largely performed in vitro, under nonphysiological conditions. We have now modified this in vitro approach to accomplish selection in growing cells. Here, we report that this new methodology has been used in vivo to select RNA elements that confer increased transcript stability. A randomized cassette was embedded in a 3'-untranslated region (UTR), downstream from the luciferase reporter open reading frame. A heterogeneous population of capped luciferase mRNA was then generated by in vitro transcription. Human liver Hep G2 cells were electroporated with this population of luciferase mRNA and total cytoplasmic RNA was isolated after varying lengths of incubation. Following RT-PCR, the 3' UTR was used to reconstruct a new population of luciferase templates, permitting subsequent cycles of in vitro transcription, electroporation, RNA isolation, and RT-PCR. Increasing the incubation time at each cycle before RNA isolation imposed selection for stable transcripts. The functional half-life of the luciferase mRNA population increased from 55 to 140 min after four cycles. Subsequent sequencing of the selected 3' UTRs revealed G-U rich elements in clones with extended chemical and functional half-lives.

3' Untranslated Regions↗

PubMed2001

In-vitro selection of highly stabilized protein variants with optimized surface.

Thermostable proteins are of prime importance in protein science, but it has remained difficult to develop general strategies for stabilizing a protein. Site-directed mutagenesis based on comparisons with thermophilic homologs is rarely successful because the sequence differences are too numerous and dominated by neutral mutations. Here we used a method of directed evolution to increase the stability of a mesophilic protein, the cold shock protein Bs-CspB from Bacillus subtilis. It differs from its thermophilic counterpart Bc-Csp from Bacillus caldolyticus at 12 surface-exposed positions. To elucidate the stabilizing potential of exposed amino acid residues, six of these variant positions were randomized by saturation mutagenesis, the corresponding library of sequences was inserted into the gene-3-protein of the filamentous phage fd, and stabilized variants were selected by the Proside technique. Proside links the increased protease resistance of stabilized protein variants with the infectivity of the phage. Many strongly stabilized variants of Bs-CspB were identified in two selections, one in the presence of a denaturant and the other at elevated temperature. Several of them are significantly more stable than the naturally thermostable homolog Bc-Csp, and the best variant reaches Tm-Csp (the homolog from the hyperthermophile Thermotoga maritima) in stability. Remarkably, this variant differs from Tm-Csp at five and from Bc-Csp at all six randomized positions. This indicates that proteins can be strongly stabilized by many different sets of surface mutations, and Proside selects them efficiently from large libraries. The course of the selection could be directed by the conditions. In an ionic denaturant non-polar surface interactions were optimized, whereas at elevated temperature variants with improved electrostatics were selected, pointing to two different strategies for stabilization at protein surfaces.

Amino Acid Sequence↗

PubMed2003

Improved catalytic efficiency and active site modification of 1,4-beta-D-glucan glucohydrolase A from Thermotoga neapolitana by directed evolution.

Thermotoga neapolitana 1,4-beta-d-glucan glucohydrolase A preferentially hydrolyzes cello-oligomers, such as cellotetraose, releasing single glucose moieties from the reducing end of the cello-oligosaccharide chain. Using directed evolution techniques of error-prone PCR and mutant library screening, a variant glucan glucohydrolase has been isolated that hydrolyzes the disaccharide, cellobiose, at a 31% greater rate than its wild type (WT) predecessor. The mutant library, expressed in Escherichia coli, was screened at 85 degrees C for increased hydrolysis of cellobiose, a native substrate rather than a chromogenic analog, using a continuous, thermostable coupled enzyme assay. The V(max) for the mutant was 108 +/- 3 units mg(-1), whereas that of the WT was 75 +/- 2 units mg(-1). The K(m) for both proteins was nearly the same. The k(cat) for the new enzyme increased by 31% and its catalytic efficiency (k(cat)/K(m)) for cellobiose also rose by 31% as compared with the parent. The nucleotide sequence of two positive clones and two null clones identified 11 single base shifts. The nucleotide transition in the most active clone caused an isoleucine to threonine amino acid substitution at position 170. Structural models for I170T and WT proteins were derived by sequence homology with Protein Data Bank code 1BGA from Paenibacillus polymyxa. Analysis of the WT and I170T model structures indicated that the substitution in the mutant enzyme repositioned the conserved catalytic residue Asn-163 and reconfigured entry to the active site.

Amino Acid Sequence↗

PubMed2005

Structural perturbation and compensation by directed evolution at physiological temperature leads to thermostabilization of beta-lactamase.

The choice of protein for use in technical and medical applications is limited by stability issues, making understanding and engineering of stability key. Here, enzyme destabilization by truncation was combined with directed evolution to create stable variants of TEM-1 beta-lactamase. This enzyme was chosen because of its implication in prodrug activation therapy, pathogen resistance to lactam antibiotics, and reporter enzyme bioassays. Removal of five N-terminal residues generated a mutant which did not confer antibiotic resistance at 37 degrees C. Accordingly, the half-life time in vitro was only 7 s at 40 degrees C. However, three cycles comprising random mutagenesis, DNA shuffling, and metabolic selection at 37 degrees C yielded mutants providing resistance levels significantly higher than that of the wild type. These mutants demonstrated increased thermoactivity and thermostability in time-resolved kinetics at various temperatures. Chemical denaturation revealed improved thermodynamic stabilities of a three-state unfolding pathway exceeding wild-type construct stability. Elongation of one optimized deletion mutant to full length increased its stability even further. Compared to that of the wild type, the temperature optimum was shifted from 35 to 50 degrees C, and the beginning of heat inactivation increased by 20 degrees C while full activity at low temperatures was maintained. We attribute these effects mainly to two independently acting boundary interface residue exchanges (M182T and A224V). Structural perturbation by terminal truncation, evolutionary compensation at physiological temperatures, and elongation is an efficient way to analyze and improve thermostability without the need for high-temperature selection, structural information, or homologous proteins.

Amino Acid Sequence↗

PubMed1997

A quantum-theoretical approach to the phenomenon of directed mutations in bacteria (hypothesis).

The Darwinian paradigm of biological evolution is based on the independence of genetic variations from selection which occurs afterwards. However, according to the phenomenon of directed mutations, some genetic variations occur mostly when the conditions favorable for their growth are created. I propose that the explanation of this phenomenon should not rely on any special 'mechanism' for the appearance of directed mutations, but rather should be based on the principles of quantum theory. I consider a physical model of adaptation whereby a polarized photon, passing through a polarizer, changes its polarization according to the angle of the polarizer. This adaptation occurs by selection of the 'fitted' polarized state which exists as a component of superposition in the initial state of the photon. However, since the same state of the incoming photon should be decomposed differently depending on the angle of the polarizer, in this case the set of variations subjected to selection depends upon the selective conditions themselves. This reveals the crucial difference between this model of adaptation and canonical Darwinian selection. Based on this analogy, the capacity of a cell to grow in particular conditions is considered an observable of the cell; the plating experiments are interpreted as measurement of this observable. The only nontrivial suggestion of the paper states that the cell, analogously to the polarized photon, may be in a state of superposition of eigenfunctions of the operator which represents this observable, and with some probability can appear as a mutant upon the measurement. Alternative growth conditions correspond to the decomposition of the same state vector into a different superposition, consistent with measurement of a different observable and appearance of different mutants. Thus, consistent with the suggested analogy, directed mutations are explained as a result of random choice from the set of outcomes determined by the environment.

Adaptation, Physiological↗

PubMed1997

Forced evolution of a regulatory RNA helix in the HIV-1 genome.

The 5'and 3'end of the HIV-1 RNA genome forms a repeat (R) element that encodes a double stem-loop structure (the TAR and polyA hairpins). Phylogenetic analysis of the polyA hairpin in different human and simian immunodeficiency viruses suggests that the thermodynamic stability of the helix is fine-tuned. We demonstrated previously that mutant HIV-1 genomes with a stabilized or destabilized hairpin are severely replication-impaired. In this study, we found that the mutant with a destabilized polyA hairpin structure is conditionally defective. Whereas reduced replication is measured in infections at the regular temperature (37 degrees C), this mutant is more fit than the wild-type virus at reduced temperature (33 degrees C). This observation of a temperature-dependent replication defect underscores that the stability of this RNA structure is critical for function. An extensive analysis of revertant viruses was performed to further improve the understanding of the critical sequence and structural features of the element under scrutiny. The virus mutants with a stabilized or destabilized hairpin were used as a starting point in multiple, independent selections for revertant viruses with compensatory mutations. Both mutants reverted to hairpins with wild-type stability along various pathways by acquisition of compensatory mutations. We identified 19 different revertant HIV-1 forms with improved replication characteristics, providing a first look at some of the peaks in the total sequence landscape that are compatible with virus replication. These experiments also highlight some general principles of RNA structure building.

Base Sequence↗

PubMed2002

Directed evolution of toluene ortho-monooxygenase for enhanced 1-naphthol synthesis and chlorinated ethene degradation.

Trichloroethylene (TCE) is the most frequently detected groundwater contaminant, and 1-naphthol is an important chemical manufacturing intermediate. Directed evolution was used to increase the activity of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 for both chlorinated ethenes and naphthalene oxidation. When expressed in Escherichia coli, the variant TOM-Green degraded TCE (2.5 +/- 0.3 versus 1.39 +/- 0.05 nmol/min/mg of protein), 1,1-dichloroethylene, and trans-dichloroethylene more rapidly. Whole cells expressing TOM-Green synthesized 1-naphthol at a rate that was six times faster than that mediated by the wild-type enzyme at a concentration of 0.1 mM (0.19 +/- 0.03 versus 0.029 +/- 0.004 nmol/min/mg of protein), whereas at 5 mM, the mutant enzyme was active (0.07 +/- 0.03 nmol/min/mg of protein) in contrast to the wild-type enzyme, which had no detectable activity. The regiospecificity of TOM-Green was unchanged, with greater than 97% 1-naphthol formed. The beneficial mutation of TOM-Green is the substitution of valine to alanine in position 106 of the alpha-subunit of the hydroxylase, which appears to act as a smaller "gate" to the diiron active center. This hypothesis was supported by the ability of E. coli expressing TOM-Green to oxidize the three-ring compounds, phenanthrene, fluorene, and anthracene faster than the wild-type enzyme. These results show clearly that random, in vitro protein engineering can be used to improve a large multisubunit protein for multiple functions, including environmental restoration and green chemistry.

Burkholderia cepacia↗

PubMed2006

Laboratory evolution of P450 BM-3 for mediated electron transfer.

Preparative synthesis with P450 monooxygenases is hampered in cell-free systems by the requirement for cofactors such as NAD(P)H as reduction equivalents. A validated medium-throughput screening system was designed for improving P450 monooxygenases by mediated electron transfer with zinc/cobalt(III)sepulchrate (Zn/Co(III)sep) as an alternative and cost-effective cofactor system. The monooxygenase P450 BM-3 F87A was used as a model system for developing the screening system in a 96-well format. A coefficient of variation of less than 10% was achieved under optimized screening conditions. The mediator evolution screen was validated by comparing the activity of P450 BM-3 to P450 BM-3 F87A and by screening a saturation mutagenesis library at amino acid position R47. For mediated electron transfer, two double mutants P450 BM-3(F87A R47F) and P450 BM-3 (F87A R47Y) were identified with a two-threefold increased catalytic efficiency (up to 32 microM(-1) min(-1) for P450 BM-3(F87A R47F) and 34 microM(-1) min(-1) for P450 BM-3 (F87A R47Y)) compared to P450 BM-3 F87A. The kinetic constants of the double mutants are, in contrast to those of P450 BM-3 F87A, dependent on Co(III)sep concentration in the presence of NADPH. kcat increases from 145 min(-1) (0.25 mM Co(III)sep) to 197 min(-1) (0.5 mM Co(III)sep), and Km decreases simultaneously from 7.0 microM to 3.7 microM, for P450 BM-3 (F87A R47F). For P450 BM-3 (F87A R47Y), kcat increases from 138 min(-1) (0.25 mM Co(III)sep) up to 187 min(-1) (0.5 mM Co(III)sep), and Km decreases from 8.2 microM to 4.2 microM. Due to lower Km values, the catalytic efficiencies were improved six times for P450 BM-3 (F87A R47F) and three times for P450 BM-3 (F87A R47Y), when comparing catalytic efficiencies of the mediated electron-transfer system to the natural reduction equivalent NADPH.

Amino Acid Substitution↗

PubMed2005

Identification of a glyphosate-resistant mutant of rice 5-enolpyruvylshikimate 3-phosphate synthase using a directed evolution strategy.

5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) is a key enzyme in the shikimate pathway and is targeted by the wide-spectrum herbicide glyphosate. Here, we describe the use of a selection system based on directed evolution to select glyphosate-resistant mutants of EPSPS. Using this system, the rice (Oryza sativa) EPSPS gene, mutagenized by Error-Prone polymerase chain reaction, was introduced into an EPSPS-deficient Escherichia coli strain, AB2829, and transformants were selected on minimal medium by functional complementation. Three mutants with high glyphosate resistance were identified in three independent glyphosate selection experiments. Each mutant contained a C(317)-->T transition within the EPSPS coding sequence, causing a change of proline-106 to leucine (P106L) in the protein sequence. Glyphosate resistance assays indicated a 3-fold increase in glyphosate resistance of E. coli expressing the P106L mutant. Affinity of the P106L mutant for glyphosate and phosphoenolpyruvate was decreased about 70-fold and 4.6-fold, respectively, compared to wild-type EPSPS. Analysis based on a kinetic model demonstrates that the P106L mutant has a high glyphosate resistance while retaining relatively high catalytic efficiency at low phosphoenolpyruvate concentrations. A mathematical model derived from the Michaelis-Menten equation was used to characterize the effect of expression level and selection conditions on kinetic (Ki and Km) variation of the mutants. This prediction suggests that the expression level is an important aspect of the selection system. Furthermore, glyphosate resistance of the P106L mutant was confirmed in transgenic tobacco (Nicotiana tabacum), demonstrating the potential for using the P106L mutant in transgenic crops.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗