Site-specific forced misincorporation mutagenesis using modified T7 DNA polymerase

A new method for forced misincorporation site-specific mutagenesisis described. The method uses an exonuclease deficient modifiedversion of T7 DNA polymerase in the presence of one dNTP toforce a misincorporation. Analysis by PAGE is used to monitorthe efficiency of such misincorporation reactions. Brief extensionof the terminally mismatched primer/template using the sameenzyme in the presence of all four dNTPs is followed by chase/ligationusing unmodified T7 DNA polymerase and T4 DNA ligase to giveheteroduplex DNA. We have applied the method to mutagenesisof the Lac Z region of M13 and found that, using strand selection,efficiencies of mutagenesis at one site are >50%. When themutating dNTP is complementary to a neighbouring homopolymerictract on the template, multiple mutation is observed and efficiencesare lower. The method is more general than internal mismatchmutagenesis and, because of its rapidity, is more expedientthan existing methods of forced misincorporation mutagenesis.     

A modified consensus approach to mutagenesis inverts the cofactor specificity of Bacillus stearothermophilus lactate dehydrogenase

Lactate dehydrogenase from Bacillus stearothermophilus is specific for NAD+. There have been several attempts to alter the cofactor specificity of this enzyme, but these have yielded enzymes with relatively low activities that still largely prefer NAD+. A modified consensus approach was used to create a library of phylogenetically preferred amino acids situated near the cofactor binding site, and variants were screened for their ability to utilize NMN+. A triple mutant (Mut31) was discovered that proved to be more catalytically efficient than wild-type. Mut31 was also better at utilizing NAD+ than the wild-type enzyme and was weakly active with NADP+ and NMN+. An analysis of single amino acid substitutions suggested that all three mutations worked in a concerted fashion to yield robust cofactor utilization. When two previously identified amino acid substitutions were introduced into the Mut31 background, the resultant quintuply substituted enzyme not only utilized NADP+ far better than the wild-type enzyme, it actually inverted its preference for NAD+ and NADP+.     

Site-directed mutagenesis to fine-tune enzyme specificity

We have used a combination of a genetic selection and oligonucleotide-directedmutagenesis to introduce a series of amino add replacementsfor a single residue into Escherichia coliglutaminyl-tRNA synthetase.The mutant enzymes mischarge supFtRNA^Tyr, with glutamine, tovarying degrees depending on the polarity of the side chainintroduced but apparently not depending on the size or shapeof the side chain. These results indicate that repulsive charge-chargeinteractions may be important for specific recognition of nucleicacids by proteins and illustrate how a mutant, derived fromgenetic selection, may be further modified in activity by oligonucleotide-directedmutagenesis.     

Recursive ensemble mutagenesis

We have developed a generally applicable experimental procedureto find functional proteins that are many mutational steps fromwild type. Optimization algorithms, which are typically usedto search for solutions to certain combinatorial problems, havebeen adapted to the problem of searching the ‘sequencespace’ of proteins. Many of the steps normally performedby a digital computer are embodied in this new molecular geneticstechnique, termed recursive ensemble mutagenesis (REM). REMuses information gained from previous iterations of combinatorialcassette mutagenesis (CCM) to search sequence space more efficiently.We have used REM to simultaneously mutate six amino acid residuesin a model protein. As compared to conventional CCM, one iterationof REM yielded a 30-fold increase in the frequency of ‘positive’mutants. Since a multiplicative factor of similar magnitudeis expected for the mutagenesis of additional sets of six residues,performing REM on 18 sites is expected to yield an exponential(30 000-fold) increase in the throughput of positive mutantsas compared to random [NN(G,C)]₁₈ mutagenesis.     

A new method for random mutagenesis of complete genes: enzymatic generation of mutant libraries in vitro

A new efficient in vitro mutagenesis method for the generationof complete random mutant libraries, containing all possiblesingle base substitution mutations in a cloned gene is described.The method is based on controlled use of polymerases. Four populationsof DNA molecules are first generated by primer elongation sothat they terminate randomly, but always just before a knowntype of base (before A, C, G or T respectively). Each of thefour populations is then mutagenized in a separate misincorporationreaction, where the correct base can now be omitted. The regenerationof wild-type sequences can thus be efficiently avoided. Also,the misincorporating nucleotide concentrations can be optimizedto give the three possible single mutations in close to equalratio. The mutagenesis can be precisely localized within a predeterminedtarget region of any size, and vector sequences remain intact.We have mutagenized the DNA coding for the -fragment of Escherichiacoli ß-galactosidase, and identified 176 differentbase substitution mutations by sequencing. The present methodgives mutant yields of 40–60%, when the mutants containabout one amino acid change per protein molecule. All typesof base substitution mutations can be generated and deletionsare rare. The efficiency of this method permits the use of relativelyelaborate screening systems to isolate mutants of either structuralgenes or regulatory regions.     

A thermostable variant of fructose bisphosphate aldolase constructed by directed evolution also shows increased stability in organic solvents

Thermostable variants of the Class II fructose bisphosphate aldolase have been isolated following four rounds of directed evolution using DNA shuffling of the fda genes from Escherichia coli and Edwardsiella ictaluri. Variants from all four generations of evolution have been purified and characterized. The variants show increased thermostability with no loss of catalytic function at room temperature. The temperature at which 50% of the initial enzyme activity is lost after incubation for 10 min (T50) of the most stable variant, 4-43D6, is increased by 11-12 degrees C over the wild-type enzymes and the half-life of activity at 53 degrees C is increased approximately 190-fold. In addition, variant 4-43D6 shows increased stability to treatment with organic solvents. DNA sequencing of the evolved variants has identified the mutations which have been introduced and which lead to increased thermostability, and the role of the mutations introduced is discussed.     

Improvement in the alkaline stability of subtilisin using an efficient random mutagenesis and screening procedure

An efficient random mutagenesis procedure coupled to a replicaplate screen facilitated the isolation of mutant subtilisinsfrom Bacillus amyloliquefaciens that had altered autolytic stabilityunder alkaline conditions. Out of about 4000 clones screened,approximately 70 produced subtilisins with reduced stability(negatives). Two dones produced a more stable subtilisin (positives)and were identified as having a single mutation, either IIe107Valor Lys2l3Arg (the wild-type amino acid is followed by the codonposition and the mutant amino acid). One of the negative mutants,Met50Val, was at a site where other homologous subtilisins containeda Phe. When the Met50Phe mutation was introduced into the B.amyloliquefaciens gene, the mutant subtilisin was more alkalinestable. The double mutant IIe107Val/Lys2l3Arg) was more stablethan the isolated single mutant parents. The triple mutant (Met50Phe/IIel07Val/Lys2l3Arg)was even more stable than IIe107Val/Lys2l3Arg (up to two timesthe autolytic half-time of wild-type at pH 12). These studiesdemonstrate the feasibility for improving the alkaline stabilityof proteins by random mutagenesis and identifying potentialsites where substitutions from homologous proteins can improvealkaline stability.     

Combination of computational prescreening and experimental library construction can accelerate enzyme optimization by directed evolution

Chiral compounds can be produced efficiently by using biocatalysts. However, wild-type enzymes often do not meet the requirements of a production process, making optimization by rational design or directed evolution necessary. Here, we studied the lipase-catalyzed hydrolysis of the model substrate 1-(2-naphthyl)ethyl acetate both theoretically and experimentally. We found that a computational equivalent of alanine scanning mutagenesis based on QM/MM methodology can be applied to identify amino acid positions important for the activity of the enzyme. The theoretical results are consistent with concomitant experimental work using complete saturation mutagenesis and high-throughput screening of the target biocatalyst, a lipase from Bacillus subtilis. Both QM/MM-based calculations and molecular biology experiments identify histidine 76 as a residue that strongly affects the catalytic activity. The experiments demonstrate its important influence on enantioselectivity.     

Improved peptide function from random mutagenesis over short 'windows'

We have applied random mutagenesis over short contiguous residuetracts (‘windows’) within an active peptide (the-peptide of ß-galactosidase) such that all windowresidues are replaced simultaneously. A novel technique usingmixed synthetic oligonucleotides and selection against an EcoKrestrictionsite has allowed the construction of libraries of mutants fortwo separate windows, sites A and B. Mutant phenotypes can beeasily assessed in vivoby a complementation test, and panelsof mutants have been quantitatively tested in vivoThis allowedthe rapid probing of structural requirements for each site.The two windows yielded markedly disparate results. Site B wasmuch less stringent in its sequence requirements for significantfunction than Site A, and mutants with improved function wereisolated at Site B alone. In addition, one Site B mutant withwild-type levels of activity showed enhanced stability to heator a protein denaturant. We propose that short tracts with thecharacteristics of Site B constitute ‘secondary’interaction sites which are more tolerant of sequence diversity.Random manipulation of such secondary sites is thus more likelyto yield up-mutations for standard or altered environments.Window mutagenesis can in principle be applied to any protein-proteinor protein-Ugand interaction.     

Directed evolution of Pseudomonas aeruginosa lipase for improved amide-hydrolyzing activity

A lipase from Pseudomonas aeruginosa was subjected to directed molecular evolution for increased amide-hydrolyzing (amidase) activity. A single round of random mutagenesis followed by screening for hydrolytic activity for oleoyl 2-naphthylamide as compared with that for oleoyl 2-naphthyl ester identified five mutants with 1.7-2.0-fold increased relative amidase activities. Three mutational sites (F207S, A213D and F265L) were found to affect the amidase/esterase activity ratios. The combination of these mutations further improved the amidase activity. Active-site titration using a fluorescent phosphonic acid ester allowed the molecular activities for the amide and the ester to be determined for each mutant without purification of the lipase. A double mutant F207S/A213D gave the highest molecular activity of 1.1 min(-1) for the amide, corresponding to a 2-fold increase compared with that of the wild-type lipase. A structural model of the lipase indicated that the mutations occurred at the sites near the surface and remote from the catalytic triad, but close to the calcium binding site. This study is a first step towards understanding why lipases do not hydrolyze amides despite the similarities to serine proteases in the active site structure and the reaction mechanism and towards the preparation of a general acyl transfer catalyst for the biotransformation of amides.     

Molecular evolution of a defined DNA sequence with accumulation of mutations in a single round by a dual approach to random chemical mutagenesis (DuARCheM)

Directed evolution has paved the way to a new era of protein and nucleic acid molecules with improved and enhanced properties. The utmost important component of directed evolution is random mutations in a defined DNA sequence. The utility of random chemical mutagenesis in directed evolution studies is dwindling due to the inherent flaws with whole-organism mutagenesis and the in vitro approach. Here, we report a novel Dual Approach to Random Chemical Mutagenesis (DuARCheM) to introduce random mutations in a defined DNA fragment. DuARCheM involves in vivo chemical mutagenesis and in vitro genetic manipulations. The resulting library revealed an accumulation of mutations in its members. These results imply that the parent mutation is carried in the further generations within the same library. This method might help to change random chemical mutagenesis because the combination of in vivo and in vitro approaches mimics the amplification and mutation that is performed by PCR-based mutagenesis, and at the same time the mutations are confined to the desired gene. Moreover, the mutagen pressure is greater in chemical mutagenesis than in a Taq-polymerase-based error-prone system. Concomitant amplification and mutation in the DuARCheM method leads to a better spectrum of mutants because the plasmid construct is exponentially amplified in the presence of mutagen pressure, unlike in the in vitro chemical mutagenesis system in which the template molecule does not replicate. This work is able to nullify all the disadvantages that are associated with random chemical mutagenesis, and could make random chemical mutagenesis an indispensable tool in directed evolution studies.     

A second-generation system for unbiased reading frame selection

Reading frame selection of nucleic acids has important implications for protein engineering and genomics. Current methods are limited because selection of the gene of interest inevitably depends on the solubility of its translated product. Here we report the construction of the pInSALect vector, which provides strict reading frame selection without concomitant selection for protein solubility or folding. This plasmid incorporates the cis-splicing VMA intein sequence from Saccharomyces cerevisiae to facilitate the post-translational self-excision of the protein of interest, thereby eliminating potential aggregation problems. Results from two libraries of chimeric glycinamide ribonucleotide formyltransferases confirm the superior performance of pInSALect over existing reading frame selection systems.     

Engineering mammalian cytochrome P450 2B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide

The previously laboratory-evolved cytochrome P450 2B1 quadruple mutant V183L/F202L/L209A/S334P (QM), which showed enhanced H(2)O(2)-mediated substrate oxidation, has now been shown to exhibit a >3.0-fold decrease in K(m,HOOH) for 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) O-deethylation compared with the parental enzyme L209A. Subsequently, a streamlined random mutagenesis and a high-throughput screening method were developed using QM to screen and select mutants with enhanced tolerance of catalytic activity to temperature and dimethyl sulfoxide (DMSO). Upon screening >3000 colonies, we identified QM/L295H and QM/K236I/D257N with enhanced catalytic tolerance to temperature and DMSO. QM/L295H exhibited higher activity than QM at a broad range of temperatures (35-55 degrees C) and maintained approximately 1.4-fold higher activity than QM at 45 degrees C for 6 h. In addition, QM/L295H showed a significant increase in T(m,app) compared with L209A. QM/L295H and QM/K236I/D257N exhibited higher activity than QM at a broad range of DMSO concentrations (2.5-15%). Furthermore, QM/K236I/D257N/L295H was constructed by combining QM/K236I/D257N with L295H using site-directed mutagenesis and exhibited a >2-fold higher activity than QM at nearly the entire range of DMSO concentrations. In conclusion, in addition to engineering mammalian cytochromes P450 for enhanced activity, directed evolution can also be used to optimize catalytic tolerance to temperature and organic solvent.     

Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide

Sequential rounds of error-prone PCR to introduce random mutationsand screenrng of the resultant mutant libraries have been usedto enhance the total catalytic activity of subtilisin E significantlyin a non-natural environment, aqueous dimethylformamide (DMF).Seven DNA substitutions coding for three new amino acid substitutionswere identified in a mutant isolated after two additional generationsof directed evolution carried out on 10M subtilisin E, previously‘evolved’ to increase its specific activity in DMF.A Bacillus subtilis-Escherichia coli shuttle vector was developedin order to increase the size of the mutant library that couldbe established in B.subtilis and the stringency of the screeningprocess was increased to reflect total as well as specific activity.This directed evolution approach has been extremely effectivefor improving enzyme activity in a non-natural environment:the resulting-evolved 13M subtilisin exhibits specific catalyticefficiency towards the hydrolysis of a peptide substrate succlnyl-Ala-Ala-Pro-Phe-p-nitroanilidein 60% DMF solution that is three times that of the parent 10Mand 471 times that of wild type subtilisin E. The total activityof the 13M culture supernatant is enhanced 16-fold over thatof the parent 10M.     

A theoretical model of restriction endonuclease NlaIV in complex with DNA, predicted by fold recognition and validated by site-directed mutagenesis and circular dichroism spectroscopy

Restriction enzymes (REases) are commercial reagents commonly used in DNA manipulations and mapping. They are regarded as very attractive models for studying protein-DNA interactions and valuable targets for protein engineering. Their amino acid sequences usually show no similarities to other proteins, with rare exceptions of other REases that recognize identical or very similar sequences. Hence, they are extremely hard targets for structure prediction and modeling. NlaIV is a Type II REase, which recognizes the interrupted palindromic sequence GGNNCC (where N indicates any base) and cleaves it in the middle, leaving blunt ends. NlaIV shows no sequence similarity to other proteins and virtually nothing is known about its sequence-structure-function relationships. Using protein fold recognition, we identified a remote relationship between NlaIV and EcoRV, an extensively studied REase, which recognizes the GATATC sequence and whose crystal structure has been determined. Using the 'FRankenstein's monster' approach we constructed a comparative model of NlaIV based on the EcoRV template and used it to predict the catalytic and DNA-binding residues. The model was validated by site-directed mutagenesis and analysis of the activity of the mutants in vivo and in vitro as well as structural characterization of the wild-type enzyme and two mutants by circular dichroism spectroscopy. The structural model of the NlaIV-DNA complex suggests regions of the protein sequence that may interact with the 'non-specific' bases of the target and thus it provides insight into the evolution of sequence specificity in restriction enzymes and may help engineer REases with novel specificities. Before this analysis was carried out, neither the three-dimensional fold of NlaIV, its evolutionary relationships or its catalytic or DNA-binding residues were known. Hence our analysis may be regarded as a paradigm for studies aiming at reducing 'white spaces' on the evolutionary landscape of sequence-function relationships by combining bioinformatics with simple experimental assays.     

酶活性设计的新方法——半理性设计

许多研究小组利用整基因随机突变形成和重组方法,已成功的进行了酶的定向进化。酶工程的最新进展就是使这些定向进化的方法相结合,并与理性酶修饰方法的要素随机结合,通过各种组合选择出适合的方法来避免定向进化和理性设计的限制。半理性方法是以多个特定的残基为靶标在结构或功能知识的基础上突变构建简洁库,它更有希望得到正确的结果。突变体的高效抽样可能对酶功能有影响,随着底物选择性和特异性的提高,及其在已知的结构中经过重新设计使酶活性有了显著提高,这种高效抽样可被引入实验,当规模较大时它会被引入计算机方法。     

A novel search method for protein sequence-structure relations using property profiles

In protein engineering and design it is very important thatresidues can be inspected in their specific environment. A standardrelational database system cannot serve this purpose adequatelybecause it cannot handle relations between individual residues.With SCAN3D we introduce a new database system for integratedsequence and structure analysis of proteins. It uses the relationalparadigm wherever possible. Its main power, however, stems fromthe ability to retrieve stretches of consecutive residues withcertain properties by comparing a property profile with allstretches of residues in the database, exploiting the orderedcharacter of proteins. In doing so, it bypasses the large numberof join operations that would be required by relational databasesystems. An additional advantage of using property profile matchingis that searches can be carried out allowing a pre-set numberof mismatches. Also, as the database is read-only, SCAN3D doesnot need interactive data update mechanisms. Queries typicalof a molecular engineering environment are demonstrated withspecific examples: analysis of peptides that induce local structure,analysis of site-dependent rotamers and residue-residue contactanalysis     

A novel mechanism of allosteric regulation of archaeal phosphoenolpyruvate carboxylase: a combined approach to structure-based alignment and model assessment

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the irreversible carboxylation of phosphoenolpyruvate (PEP) and plays a crucial role in fixing atmospheric CO(2) in C(4) and CAM plants. The enzyme is widespread in plants and bacteria and mostly regulated allosterically by both positive and negative effectors. Archaeal PEPCs (A-PEPCs) have unique characteristics in allosteric regulation and molecular mass, distinct from their bacterial and eukaryote homologues, and their amino acid sequences have become available only recently. In this paper, we generated a structure-based alignment of archaeal, bacterial and eukaryote PEPCs and built comparative models using a combination of fold recognition, sequence and structural analysis tools. Our comparative modeling analysis identified A-PEPC-specific strong interactions between the two loops involved in both allostery and catalysis, which explained why A-PEPC is not influenced by any allosteric activators. We also found that the side-chain located three residues before the C-terminus appears to play a key role in determining the sensitivity to allosteric inhibitors. In addition to these unique features, we revealed how archaeal, bacterial and eukaryote PEPCs would share a common catalytic mechanism and adopt a similar mode of tetramer formation, despite their divergent sequences. Our novel observations will help design more efficient molecules for ecological and industrial use.     

A novel approach for albumin determination in aqueous media by using temperature‐ and pH‐sensitive N‐isopropylacrylamide‐co‐N‐[3‐(dimethylamino)‐propyl]methacrylamide random copolymers

Random copolymers of N‐isopropylacrylamide (NIPA) and N‐[3‐(dimethylamino)propyl]methacrylamide (DMAPM) were synthesized by solution polymerization using azobisizobutyronitrile as the initiator in 1,4‐dioxane at 60°C. NIPA‐co‐DMAPM copolymer exhibited both temperature and pH sensitivity. Thermally reversible phase transitions were observed both in the acidic and the alkaline pH regions for copolymers produced with different DMAPM/NIPA feed ratios. The pH dependency of the lower critical solution temperature (LCST) was stronger for copolymers produced with higher DMAPM feed concentrations. NIPA‐co‐DMAPM random copolymer was also sensitive to the albumin concentration. In the presence of albumin, thermally irreversible phase transitions were observed in slightly acidic and neutral media. However, reversible transitions were obtained in aqueous media containing albumin at basic pH. The phase‐transition temperature of NIPA‐co‐DMAPM copolymer significantly decreased with increasing albumin concentration at both acidic and alkaline pH values. This behavior was explained by albumin binding onto the copolymer chains by means of H‐bond formation between the dimethylamino groups of the copolymer and the carboxyl groups of albumin. For a certain range of albumin concentration, the phase‐transition temperature exhibited a linear decrease with increasing albumin concentration. By utilizing this behavior, a simple albumin assay was developed. The results indicated that NIPA‐co‐DMAPM copolymer could be utilized as a new reagent for the determination of albumin concentration in the aqueous medium. The proposed method was valid for the albumin concentration range of 0–4000 μg/mL. The protein concentrations commonly utilized in biotechnological studies fall in the range of the proposed method. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 84: 2060–2071, 2002; DOI 10.1002/app.10503