An attempt to unify the structure of polymerases

With the great availability of sequences from RNA- and DNA-dependentRNA and DNA polymerases, it has become possible to delineatea few highly conserved regions for various polymerase types.In this work a DNA polymerase sequence from bacteriophage SPO2was found to be homologous to the polymerase domain of the Klenowfragment of polymerase I from Escherichia coli, which is knownto be closely related to those from Staphylococcus pneumoniae,Thermits aquaticus and bacteriophages T7 and T5. The alignmentof the SPO2 polymerase with the other five sequences considerablynarrowed the conserved motifs in these proteins. Three of themotifs matched reasonably all the conserved motifs of anotherDNA polymerase type, characterized by human polymerase a. Itis also possible to find these three motifs in monomeric DNA-dependentRNA polymerases and two of them in DNA polymerase ßand DNA terminal transferases. These latter two motifs alsomatched two of the four motifs recently identified in 84 RNA-dependentpolymerases. From the known tertiary architecture of the Klenowfragment of E.coli pol I, a spatial arrangement can be impliedfor these motifs. In addition, numerous biochemical experimentssuggesting a role for the motifs in a common function (dNTPbinding) also support these inferences. This speculative hypothesis,attempting to unify polymerase structure at least locally, ifnot globally, under the pol I fold, should provide a usefulmodel to direct mutagenesis experiments to probe template andsubstrate specificity in polymerases.     

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.     

Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling

The possibility of increasing the affinity of a Taq DNA polymerasespecific binding protein (affibody) was investigated by an -helixshuffling strategy. The primary affibody was from a naive combinatoriallibrary of the three-helix bundle Z domain derived from staphylococcalprotein A. A hierarchical library was constructed through selectivere-randomization of six amino acid positions in one of the two-helices of the domain, making up the Taq DNA polymerase bindingsurface. After selections using monovalent phage display technology,second generation variants were identified having affinities(K_D) for Taq DNA polymerase in the range of 30–50 nM asdetermined by biosensor technology. Analysis of binding dataindicated that the increases in affinity were predominantlydue to decreased dissociation rate kinetics. Interestingly,the affinities observed for the second generation Taq DNA polymerasespecific affibodies are of similar strength as the affinitybetween the original protein A domain and the Fc domain of humanimmunoglobulin G. Further, the possibilities of increasing theapparent affinity through multimerization of affibodies wasdemonstrated for a dimeric version of one of the second generationaffibodies, constructed by head-to-tail gene fusion. As comparedwith its monomeric counterpart, the binding to sensor chip immobilizedTaq DNA polymerase was characterized by a threefold higher apparentaffinity, due to slower off-rate kinetics. The results showthat the binding specificity of the protein A domain can bere-directed to an entirely different target, without loss ofbinding strength.     

A thermoresistant mutant of ribonuclease T1 having three disulfide bonds

Molecular-dynamic calculations predict that, if Tyr24 and Asn84are each replaced by a Cys residue, it should be possible toform a third disulfide bond in ribonuclease T1 (RNase T1) betweenthese residues, with only minimal conformational changes atthe catalytic site. The gene encoding such a mutant variantof RNase T1 (Tyr24 – Cys24, Asn84 – Cys84) was constructedby the cassette mutagenesis method using a chemically synthesizedgene. In order to reduce the toxic effect of the mutant enzyme(RNase T1S) on an Escherichia coli host, we arranged for theprotein to be secreted into the periplasmic space by using avector that harbors a gene for an alkaline phosphatase signalpeptide under the control of the trp promoter. The nucleolyticactivity of RNase T1S toward pGpC was approximately the sameas that of RNase T1 at 37°C (pH 7.5). Moreover, at 55°C,RNase T1S retained nearly 70% of its activity while the activityof the wild-type enzyme was reduced to <10%. RNase T1S wasalso more resistant to denaturation by urea than the wild-typeenzyme. However, unlike RNase T1, RNase T1S was irreversiblyand almost totally inactivated by boiling at 100°C for 15min.     

A novel random mutagenesis approach using human mutagenic DNA polymerases to generate enzyme variant libraries

The in vitro MutaGen procedure is a new random mutagenesis method based on the use of low-fidelity DNA polymerases. In the present study, this technique was applied on a 2 kb gene encoding amylosucrase, an attractive enzyme for the industrial synthesis of amylose-like polymers. Mutations were first introduced during a single replicating step performed by mutagenic polymerases pol beta and pol eta. Three large libraries (>10(5) independent clones) were generated (one with pol beta and two with pol eta). The sequence analysis of randomly chosen clones confirmed the potential of this strategy for the generation of diversity. Variants generated by pol beta were 4-7-fold less mutated than those created with pol eta, indicating that our approach enables mutation rate control following the DNA polymerase employed for mutagenesis. Moreover, pol beta and pol eta provide different and complementary mutation spectra, allowing a wider sequence space exploration than error-prone PCR protocols employing Taq polymerase. Interestingly, some of the variants generated by pol eta displayed unusual modifications, including combinations of base substitutions and codon deletions which are rarely generated using other methods. By taking advantage of the mutation bias of naturally highly error-prone DNA polymerases, MutaGen thus appears as a very useful tool for gene and protein randomisation.     

Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli

For the first time the pro-form of a recombinant cysteine proteinasehas been expressed at a high level in Escherichia coli. Thisinactive precursor can subsequently be processed to yield activeenzyme. Sufficient protein can be produced using this systemfor X-ray crystallographic structure studies of engineered proteinases.A cDNA clone encoding propapain, a precursor of the papaya proteinase,papain, was expressed in E.coli using a T7 polymerase expressionsystem. Insoluble recombinant protein was solubilized in 6 Mguanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6.A protein-glutathione mixed disulphide was formed by dilutioninto oxidized glutathione and 6 M GuHCl, also at pH 8.6. Finalrefolding and disulphide bond formation was induced by dilutioninto 3 mM cysteine at pH 8.6. Renatured propapain was processedto active papain at pH 4.0 in the presence of excess cysteine.Final processing could be inhibited by the specific cysteineproteinase inhibitors E64 and leupeptin, but not by pepstatin,PMSF or EDTA. This indicates that final processing was due toa cysteine proteinase and suggests that an autocatalytic eventis required for papain maturation.     

Lysines 72, 80 and 213 and aspartic acid 210 of the Lactococcus lactis LacR repressor are involved in the response to the inducer tagatose-6-phosphate leading to induction of lac operon expression

Site-directed mutagenesis of the Lactococcus lactis lacR genewas performed to identify residues in the LacR repressor thatare involved in the induction of lacABCDFEGX operon expressionby tagatose-6-phosphate. A putative inducer binding domain locatednear the C-terminus was previously postulated based on homologystudies with the Escherichia coli DeoR family of repressors,which all have a phosphorylated sugar as inducer. Residues withinthis domain and lysine residues that are charge conserved inthe DeoR family were changed into alanine or arginine. The productionof the LacR mutants K72A, K80A, K80R, D210A, K213A and K213Rin the LacR-deficient L.lactis strain NZ3015 resulted in repressedphospho-ß-galactosidase (LacG) activities and decreasedgrowth rates on lactose. Gel mobility shift assays showed thatthe complex between a DNA fragment carrying the lac operatorsand LacR mutants K72A, K80A, K213A and D210A did not dissociatein the presence of tagatose-6-phosphate, in contrast to wildtype LacR. Other mutations (K62A/K63A, K72R, K73A, K73R, T212A,F214R, R216R and R216K) exhibited no gross effects on inducerresponse. The results strongly suggest that the lysines at positions72, 80 and 213 and aspartic acid at position 210 are involvedin the induction of lac operon expression by tagatose-6-phosphate.     

A method for construction of long randomized open reading frames and polypeptides

A method is presented for construction of randomized open readingframe sequences (ORFs) and gene libraries containing them. Thebuilding blocks for the ORFs were 75 bp long DNA fragments generatedby cloning sequences from a single synthetic oligonucleotidepreparation by bridge mutagenesis. The fragments had the propertythat, regardless of their orientation in the ligated product,the ORF of the construct was maintained. The heterogeneity ofthe ORFs resulted from the random ligation of 2000 differentDNA fragments. The randomized ORFs were cloned downstream fromthe lac promoter in a multicopy plasmid in Escherichia coli.To test the method, a library of 10⁶ clones was constructed.     

Replacing the glutamate ligand in the structural zinc site of Sulfolobus solfataricus alcohol dehydrogenase with a cysteine decreases thermostability

The alcohol dehydrogenase gene from the thermophilic archaeumSulfolobus solfataricus has been subcloned and expressed inEscherichia coli under the control of the T7 inducible promoter.Therecombinant protein shows properties analogous to those of thenative enzyme, including thermostability, despite the fact thatE.coli does not post-translationally modify two lysine residueswhich are N--methylated in the native enzyme. We constructeda 3-D model of the S.solfataricus alcohol dehydrogenase usingthe known structure of its isozyme from horse liver as a template.Our analysis of the structural zinc binding site suggested thatthis site is present andfunctional in the S.solfataricus enzymeand that a glutamate ligand can contribute to thermostabilityby influencing electrostatic interactions around the metal centre.To investigate thishypothesis, we constructed, expressed andcharacterized a mutant where the glutamate is replaced by acysteine, thus restoring the zinc binding site of mesophilicalcohol dehydrogenases. Themutant shows the same activity buta reduced thermostability with respect to the wild-type recombinantprotein, as suggested by our model.     

The amino acid sequence required for 5' -> 3' exonuclease activity of Bacillus caldotenax DNA polymerase

We studied the 5' 3' exonuclease activity of Bacillus caldotenaxDNA polymerase by site-directed mutagenesis. Among seven mutantsconstructed, two mutant DNA polymerases with an amino acid substitutionof Glyl84 Asp or Glyl92 Asp were confirmed to be deficientin this exonuclease. The two positions corresponded to thoseof the Escherichia coli DNA polymerase I mutants defective in5' 3' exonuclease, polA480ex and polA214. These results provideexperimental support for the proposed amino acid sequence essentialfor the 5' 3' exonuclease activity associated with eubacterialpolymerase I–like DNA polymerases (family A), includingE.coli and Thermits aquaticus.     

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.     

Monte Carlo docking of protein-DNA complexes: incorporation of DNA flexibility and experimental data

A Monte Carlo simulation program (MONTY) has been developedto dock proteins onto DNA. Protein and DNA interact via square-wellpotentials for hydrogen bond and van der Waals interactions.The effect of the inclusion of DNA flexibility and experimentallyderived restraints has been tested on members of the helix-turn-helixfamily of DNA binding proteins. Unwinding and bending the DNAdouble helix improves the number of correctly retrieved hydrogenbonds in simulations starting from the 434 cro protein monomercomplexed with a standard B-DNA O_Rl half-site. Agreement withphosphate ethylation interference and mutagenesis data is rewardedwith energy bonuses. This protocol was tested on protein-DNAcomplexes of 434 cro, lac headpiece and a mutant lac headpieceresembling the gal repressor headpiece with the recognitionhelices in correct and reversed orientations in the DNA majorgroove. The inclusion of experimental data gives an improvedconvergence of the correctly oriented structures and allowsfor an easier discrimination between correctly and incorrectlydocked complexes     

Three-dimensional structures of chimeric enzymes between Bacillus subtilis and Thermus thermophilus 3-isopropylmalate dehydrogenases

The 3-D structures of two chimeric enzymes (4M6T and 2T2M6T)between the Bacillus subtilis and Thermus thermophilus 3-isopropylmalatedehydrogenases were analysed by X-ray diffraction in order toinvestigate their different thermostabilities. The structureof 2T2M6T was determined by the difference Fourier method andthat of 4M6T by rigid body refinement, as based on the structureof the T. thermophilus enzyme. These structures were refinedstereochemically to an R-factor of 0.193 at 2.5 Å resolutionfor 4M6T and to an R-factor of 0.195 at 2.2 Å resolutionfor 2T2M6T. The 3-D structures of 4M6T and 2T2M6T were veryclose to the structure of the T.thermophilus enzyme, conspicuousdifferences being at the molecular surface, In particular, 2T2M6Thaving a larger reduction in thermostability was more closelyrelated to the T.thermophilus enzyme. However, their correlationsbetween C-atom displacements and the root squares of the temperaturefactors were significantly different from each other.     

Over-production of 5-enolpyruvylshikimate-3-phosphate synthase in Escherichia coli: use of the T7 promoter

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, the productof the Escherichia coli aroA gene, has been overproduced inE.coli BL21(DE3) under the control of the T7 gene 10 promoterand ribosome binding site, to a level of {small tilde}50% oftotal cell protein. EPSP synthase is the primary target of thepost-emergence herbicide, glyphosate, commonly known as Roundup^TM.A simple two step purification is described, which results in99% pure homogeneous protein (as determined by PAGE). The integrityof the protein has been compared with previously characterizedprotein from .E.coli AB2829(pKD501) by determination of itskinetic parameters, N-terminal protein and DNA sequences, aminoacid analysis and ¹³C-NMR spectroscopy. This new overproducingstrain readily provides the gram quantities of highly pure proteinrequired for NMR studies of the active site and the developmentof novel time-resolved solid-state NMR techniques currentlyunderway in this laboratory.     

The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease

Cavities in the hydrophobic core of the neutral protease ofBacillus stearothermophilus were analyzed using a threedimensionalmodel that was inferred from the crystal structure of thermolysin,the highly homologous neutral protease of B.thermoproteolyticus(85% sequence identity). Site–directed mutagenesis wasused to fill some of these cavities, thereby improving hydrophobicpacking in the protein interior. The mutations had small effectson the thermostability, even after drastic changes, such asLeu284Trp and Met168Trp. The effects on T50, the temperatureat which 50% of the enzyme is irreversibly inactivated in 30min, ranged from 0.0 to +0.4°C. These results can be explainedby assuming that the mutations have positive and negative structuraleffects of approximately the same magnitude. Alternatively,it could be envisaged that the local unfolding steps, whichrender the enzyme susceptible towards autolysis and which arerate limiting in the process of thermal inactivation, are onlyslightly affected by alterations in the hydrophobic core.     

A rapid and effective procedure for screening protease mutants

We describe a simple and effective procedure to screen for activeproteases among a large number of mutants. First, the mutantsare genetically tested by the protease activity produced inthe periplasm of transformed bacteria which supplies the cellswith a nitrogen source by hydrolyzing a protein applied to plates.Then a less sensitive activity staining and an X-ray film digestionassay are used to verify and estimate the activity of the mutantsthat proved to be positive in the first step. Depending essentiallyon the level of periplasmic protease activity, the method candetect both the activity and the stability of the expressedenzymes. We calibrated the method with transformants that producewild-type trypsin, chymotrypsin and trypsin mutants of knownactivity. Using this method we found two active revertants ofthe inactive Asn102 trypsin mutant, by screening {small tilde}4.4x10⁴random mutants that were generated by the polymerase chain reactionon a cDNA fragment. This procedure should be useful in searchingfor proteases of novel specificity and/or reaction chemistryengineered by random mutagenesis, and also for in vitro evolutionstudies.     

Altering substrate preference of carboxypeptidase Y by a novel strategy of mutagenesis eliminating wild type background

To change the substrate preference of carboxypeptidase Y theputative substrate binding pocket was subjected to random mutagenesis.Based upon the three-dimensional structure of a homologous enzymefrom wheat, we hypothesized that Tyr147, Leu178, Glu215, Arg216,Ile340 and Cys341 are the amino acid residues of carboxypeptidaseY that constitute S₁ the binding pocket for the penultimateamino acid side chain of the substrate. We developed a new andgenerally applicable mutagenesis strategy to facilitate efficientscreening of a large number of mutants with multiple changesin carboxypeptidase Y. The key feature is the elimination ofwild type background by introducing a nonsense codon at eachtarget site for subsequent mutagenesis by degenerate oligonucleotides.The entire hypothesized S₁ binding pocket and subsets of itwere subjected to saturation mutagenesis by this strategy, andscreening yielded a number of mutant enzymes which have up to150 times more activity (k_cat/K_m towards CBZ-LysLeu-OH thanthe wild type enzyme. All selected mutants with increased activityhave mutations at position 178. Mutagenesis of positions 215and 216 has virtually no effect on the activity, while mutatingpositions 340 and 341 generally reduces activity.     

The Escherichia coli aspartate receptor: sequence specificity of a transmembrane helix studied by hydrophobic-biased random mutagenesis

The Escherichia coli aspartate receptor is a dimer with twotransmembrane sequences per monomer that connect a periplasmicligand binding domain to a cytoplasmic signaling domain. Themethod of 'hydrophobic-biased' random mutagenesis, that we describehere, was used to construct mutant aspartate receptors in whicheither the entire transmembrane sequence or seven residues nearthe center of the transmembrane sequence were replaced withhydrophobic and polar random residues. Some of these receptorsresponded to aspartate in an in vivo chemotaxis assay, whileothers did not. The acceptable substitutions included hydrophobicto polar residues, small to larger residues, and large to smallerresidues. However, one mutant receptor that had only a few hydrophobicsubstitutions did not respond to aspartate. These results addto our understanding of sequence specificity in the transmembraneregions of proteins with more than one transmembrane sequence.This work also demonstrates a method of constructing familiesof mutant proteins containing random residues with chosen characteristics.