A single amino acid substitution can restore the solubility of aggregated colicin A mutants in Escherichia coli

Mutants of colicin A have been prepared in which the three tryptophanresidues (Trp86, Trpl30 and Trpl40), localized in the C-terminaldomain of the soluble wild-type protein, have been substitutedby phenylalanine. The Trpl40Phe single mutation had the effectof decreasing the percentage of protein that is expressed asinsoluble aggregates. The created hydrophobic cavity decreasedthe stability of the protein during its folding, resulting inpartial aggregation in the cytoplasm of the Escherichia coli-producingcells. Aggregation was increased when Trpl40 was substitutedby Lys, Leu or Cys, or if the Trpl40 mutation was combined withthe Trp86Phe and/ or Trpl30Phe mutations. A single mutation,Lysll3Phe, however, was able to restore the solubility of theaggregated mutants in vivo. Detailed analysis of the 3-D structureof the C-terminal domain of colicin A suggests that fillingof the hydrophobic cavity is responsible for this effect.     

Cumulative stabilizing effects of hydrophobic interactions on the surface of the neutral protease from Bacillus subtilis

Using genetically engineered mutants of the neutral pro-teasefrom Bacillus stearothermophilus (BsteNP), it had been shownthat the surface-exposed structural motif constituted by Phe63embedded in a four amino acid hydrophobic pocket is criticalfor the thermal stability of the thermophilic neutral proteasesfrom Bacilli. To measure the stabilizing contribution of eachhydrophobic interaction taking place between Phe63 and the hydrophobicpocket, we grafted this structural motif in the neutral proteasefrom the mesophile Bacillus subtilis (BsubNP). This was accomplishedby first creating the Thr63Phe mutant of BsubNP and then generatinga series of mutants in which the four amino acids which in thermolysinsurround Phe63 and form the hydrophobic pocket were added oneafter the other. By analysing the thermal stability of eachmutant it was found that the 2°C destabilizing effect ofthe Thr63Phe substitution was completely suppressed by the additionof the four amino acid hydrophobic pocket, each replacementproviding a stabilizing contribution of approxi mately 0.8–1°C.These results are discussed in the light of the peculiar mechanismof thermal inactivation of proteolytic enzymes.     

Alteration of catalytic properties of chymosin by site-directed mutagenesis

Artificial mutations of chymosin by recombinant DNA techniqueswere generated to analyze the structure–function relationshipin this characteristic aspartk proteinase. In order to preparethe mutant enzymes in their active form, we established proceduresfor purification of correctly refolded prochymosin from inclusionbodies produced in Escherichia coli transformants and for itssubsequent activation. Mutagenesis by linker insertion intocDNA produced several mutants with an altered ratio of milkclotting activity to proteolytic activity and a different extentof stability. In addition to these mutants, several mutantswith a single amino acid exchange were also constructed by site-directedmutagenesis and kinetic parameters of these mutant enzymes weredetermined by using synthetic hexa- and octa-peptides as substrates.Exchange of Tyr75 on the flap of the enzyme to Phe caused amarked change of substrate specificity due to the change ofk_cat or K_m, depending on the substrate used. Exchange of Val110and Phe111 also caused a change of kinetic parameters, whichindicates functional involvement of these hydrophobic residuesin both the catalytic function and substrate binding. The mutantLys220–Leu showed a marked shift of the optimum pH tothe acidic side for hydrolysis of acid-denatured haemoglobinalong with a distinct increase in k_cat for the octa-peptidein a wide pH range.     

The function of the Saccharomyces cerevisiae iso-1-cytochrome c gene is independent of the codon at invariant residue Phe82 when the gene is present on a low-copy-number vector

Phe82 is the most studied invariant residue of cytochrome c.However, the physiological relevance of amino acid substitutionsat this position is unclear because previous studies were eitherperformed in vitro (i.e. using purified protein) or in yeastwhere the gene for the protein is present on a multi-copy vector.Multi-copy vectors yield a level of cytochrome c in yeast thatis greater than the wild-type level. Oligodeoxyribonucleotide-directedmutagenesis was used to change the codon for Phe82 to that ofthe other 19 naturally occurring amino acids as well as theamber stop codon. The alleles are present on a yeast shuttlephagemid containing the CEN6 gene which ensures a vector copynumber of one to two in yeast. All the missense alleles supportgrowth under conditions requiring a functional iso-1-cytochromec. However the F82C, F82P, and F82R variants grow at a significantlylower rate. After selection for function, phagemids were rescuedfrom the transformants and the identity of the mutation verified.It is concluded that all 20 amino acids are capable of supportingfunction. Reasons for the evolutionary invariance of Phe82 arediscussed.     

Protein engineering of alcohol dehydrogenases; effects of amino acid changes at positions 93 and 48 of yeast ADH1

By protein engineering we have investigated changes to two aminoacid residues (Trp93 and Ser48) in the substrate pocket of yeastalcohol dehydrogenase 1. Upon changing Thr48 to serine we producedan enzyme which has markedly greater activity towards aliphaticalcohols with chain length up to 8, together with a generalincrease in catalytic activity (V/K). Changes at position 93were less pronounced, with the Phe enzyme being more activethan the parent towards the range of alcohols but with the alanineenzyme showing very little difference from the wild-type. Enzymeswith the double changes at 48 and 93 showed increased activitytowards alcohols with 3–8 carbons but the increases werenot additive over the single changes. The enzymes with changesat the two positions would metabolize both stereoisomers of2-octanol whereas the parent ADH would attack only one of them.None of the engineered enzymes would attack cyclohexanol oraromatic alcohols. The results are in general agreement withthe prediction that reducing the size of amino acids in thesubstrate pocket would enhance the ability to oxidize alcoholslarger than ethanol.     

Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl--disulfide interchange

In the preceding paper in this issue, we described the overproduction of one mutant chicken lysozyme in Escherichia coil.Since this lysozyme contained two amino acid substitutions (Ala³¹ValandAsn¹⁰⁶Ser)in addition to an extra methionine residue at theNH₂-terminus the substituted amino acid residues were convertedback to the original ones by means of oligonucleotide-directedsite-specific mutagenesis and in vitro recombination. Thus fourkinds of chicken lysozyme [Met^–1 Val³¹Ser¹⁰⁶-, Met^–1Ser¹⁰⁶-,Met^–1 Val³¹-and Met^–1 (wild type)] wereexpressed in E. coli. From the results of folding experimentsof the reduced lysozymes by sulfhydryl-disulfide interchangeat pH 8.0 and 38°C, follow ed by the specific activity measurementsof the folded en zymes, the following conclusions can be drawn:(i) an extra methionine residue at the NH₂-terminus reducesthe folding rate but does not affect the lysozyme activity ofthe folded enzyme; (ii) the substitution of Asn¹⁰⁶ by Ser decreasesthe activity to 58% of that of intact native lysozyme withoutchanging the folding rate; and (iii) the substitution of Ala³¹Val prohibits the correct folding of lysozyme. Since the wildtype enzyme (Met^–1-lysozyme) was activated in vitro withoutloss of specific activity, the systems described in this study(mutagenesis, overproduction, purification and folding of inactivemutant lysozymes) may be useful in the study of folding pathways,expression of biological activity and stability of lysozyme.     

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.     

Isolation and characterization of mutants of firefly luciferase which produce different colors of light

The luciferase cDNA from the ‘Genji’ firefly, Luciolacruciata, was mutated with hydroxylamine to isolate mutant lucierases.Some of the isolated mutant enzymes produced different colorsof light, ranging from green to red. Five such mutants, producinggreen (_max = 558 nm), yellow-orange (_max = 595 nm), orange (_max= 607 nm) and red light (_max = 609 and 612 nm), were analyzed.The mutations were found to be single amino acid changes, fromVal239 to IIe, Pro452 to Ser, Ser286 to Asn, Gly326 to Ser andHis433 to Tyr respectively.     

Involvement of a residue at position 75 in the catalytic mechanism of a fungal aspartic proteinase, Rhizomucor pusilus pepsin. Replacement of tyrosine 75 on the flap by asparagine enhances catalytic efficiency

Residue 75 on the flap, a beta hairpin loop that partially coversthe active site cleft, is tyrosine in most members of the asparticproteinase family. Site-directed mutagenesis was carried outto investigate the functional role of this residue in Rhizomucorpusilus pepsin, an aspartic proteinase with high milk-clottingactivity produced by the fungus Rhizomucor pusillus. A set ofmutated enzymes with replacement of the amino acid at position75 by 17 other amino acid residues except for His and Gly wasconstructed and their enzymatic properties were examined. Strongactivity, higher than that of the wild-type enzyme, was foundin the mutant with asparagine (Tyr75Asn), while weak but distinctactivity was observed in Tyr75Phe. All the other mutants showedmarkedly decreased or negligible activity, less than 1/1000of that of the wild-type enzyme. Kinetic analysis of Tyr75Asnusing a chromogenic synthetic oligopeptide as a substrate revealeda marked increase in k_cat with slight change in K_m, resultingin a 5.6-fold increase in k_cat/k_m. When differential absorptionspectra upon addition of pepstatin, a specific inhibitor foraspartic proteinase, were compared between the wild-type andmutant enzymes, the wild-type enzyme and Tyr75Asn, showing strongactivity, had spectra with absorption maxima at 280, 287 and293 nm, whereas the others, showing decreased or negligibleactivity, had spectra with only two maxima at 282 and 288 nm.This suggests a different mode of the inhibitor binding in thelatter mutants. These observations suggest a crucial role ofthe residue at position 75 in enhancing the catalytic efficiencythrough affecting the mode of substrate-binding in the asparticproteinases.     

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.     

Conformational stability of human skeletal tropomyosins modified by site-directed mutagenesis

We have used human ß-tropomyosin produced in Escherichiacoli and deletion mutants obtained by site-directed mutagenesisto analyse the conformational stability of this molecule undervarious experimental conditions. Protein engineering has allowedus to answer some questions raised by stability analysis ofthe wild-type tropomyosin. The complex pattern of denaturationis due neither to heterogeneity of the preparation nor to head-to-tailinteractions. The N- and C-termini are not of importance forthe thermal stability of the molecule. On the contrary, deletionof the 31 C-terminus amino acids leads to a dramatic decreaseof the stability observed in guanidinium chloride. This loweringis interpreted as the participation of one more guanidiniumchloride ions to the denaturation equilibrium. Analysis of thestability in presence of organic solvents reveals that acetonitrileand methanol induce opposite effects. Investigation of theseeffects by three methods (CD, fluorescence and electrophoresisthat measure respectively the content in alpha-helix, the contactbetween the two strands and the strands exchange) leads to theconclusion that strand separation can precede the denaturationof the alpha-helix.     

Selective proton labelling of amino acids in deuterated bovine calbindin D9K. A way to simplify 1H-NMR spectra

A technique for proton labelling of selected amino acids indeuterated calbindin D_9K, heterologously expressed in E.coli,was developed in order to simplify and obtain higher resolutionin ¹H-NMR spectra. The spectra from two double-labelling experiments,Val plus Ser and Val plus Leu, when compared to the uniformlyprotonated protein showed a dramatically simpler pattern withlow background signals and gave considerably sharper resonancesdue to reduced relaxation rates in the deuterated proteins.The selective proton labelling technique will enable detailedand rapid analysis of interesting domains of proteins and willalso make the analysis of larger proteins feasible.     

Thermosensitive mutants of Aspergillus awamori glucoamylase by random mutagenesis: inactivation kinetics and structural interpretation

Abstract Seven thermosensitive glucoamylase mutants generated by randommutagenesis and expressed inSaccharomyces cerevisiae were sequencedand their inactivation kinetics were determined. Wild-type glucoamylaseexpressed in S.cerevisiae was more glycosylated and more stablethan the native Aspergillus niger enzyme. All mutants had lowerfree energies of inactivation than wild-type glucoamylase. Inthe Ala39 Val, Ala302 Val and Leu410 Phe mutants, small hydrophobicresidues were replaced by larger ones, showing that increasesin size and hydrophobicity of residues included in hydrophobicclusters were destabilizing. The Gly396 Ser and Gly407 Aspmutants had very flexible residues replaced by more rigid ones,and this probably induced changes in the backbone conformationthat destabilized the protein. The Prol28 Ser mutation changeda rigid residue in an a-helix to a more flexible one, and destabilizedthe protein by increasing the entropy of the unfolded state.The Ala residue in the Ala442 Thr mutation is in the highlyO-glycosylated region surrounded by hydrophilk residues, whereitmay be a hydrophobic anchor Unking the O-glycosylated arm tothe catalytic core. It was replaced by a residue that potentiallyis O-glycosylated. In five of the seven mutations, residuesthat were part of hydrophobic microdomains were changed, confirmingthe importance of the latter in protein stability and structure     

Structure--activity relationships in human interleukin-1{alpha}: identification of key residues for expression of biological activities

To identify the sites important for the different biologicalactivities of human interleukin-l (hIL-1), 56 single-amino acid-substitutedmutants of hIL-l were produced in Escherichia coli using site-directedmutagenesis, and were examined for their biological activitiessuch as mouse lymphocyte activating factor activity (LAF activity),cytostatic activity against human melanoma cells A-375 (A375activity) and prostaglandin E2 (PGE2) inducing activity in humanosteosarcoma cells MG-63 (PEI activity). Two amino acid residues,Asp26 and Asp151, were found to be important for these activities.The replacement of Asp26 by Val caused a decrease in LAF andA375 activities by one or two orders of magnitude and a slightdecrease in A375 activity. The Tyr or Phe substitution for Asp151caused decreases in LAF and A375 activities by one or two ordersof magnitude and complete loss of PEI activity. The change fromAsp151 to Lys or Arg resulted in marked decrease in LAF activityand complete loss of A375 and PEI activities. Since Asp26 andAsp151 are close to each other in the three-dimensional structure,the region involving these amino acids seems to be importantfor the biological activities of hIL-1.     

Identification of two amino acids contributing the high enzyme activity in the alkaline pH range of an alkaline endoglucanase from a Bacillus sP

An alkaline cellulase ß-1,4-endoglucanase; NK1) froman alkalophilk Bacillus sp. shows great similarity in aminoacid sequence to a neutral cellulase (BSC) from Bacillus subtilis,despite a considerable difference in their pH activity profiles.Multiple amino acid exchanges by site-directed mutagenesis,using BSC as the reference, were performed on the residues inregion 5 of NK1, which was previously shown to be responsiblefor the high enzyme activity of this alkaline cellulase in abroad alkaline pH range. Two amino acid residues, Ser287 andAla296, were identified as being responsible for the activityin the alkaline range. The double mutation, Ser287 to Asn andAla296 to Ser, of NK1 made its pH activity profile almost thesame as that of BSC. On the other hand, the pH activity profilein the acidic range was not significantly affected by variousamino acid replacements including these two positions in region5. This observation, together with the information availableon other endoglucanases, suggests that the above two amino acidsubstitutions caused a profound effect through rearrangementof the hydrogen bond network forming the substrate-binding siteor the catalytic site.     

Probing the role of threonine and serine residues of E.coli asparaginase II by site-specific mutagenesis

Site-specific mutagenesis has been used to probe amino acidresidues proposed to be critical in catalysis by Escherichiacoli asparaginase II. Thr12 is conserved in all known asparaginases.The catalytic constant of a T12A mutant towards L-aspartk acidß-hydroxamate was reduced to 0.04% of wild type activity,while its An, and stability against urea denaturation were unchanged.The mutant enzyme T12S exhibited almost normal activity butaltered substrate specificity. Replacement of Thr119 with Alaled to a 90% decrease of activity without markedly affectingsubstrate binding. The mutant enzyme S122A showed normal catalyticfunction but impaired stability in urea solutions. These dataindicate that the hydroxyl group of Thr12 is directly involvedin catalysis, probably by favorably interacting with a transitionstate or intermediate. By contrast, Thr119 and Ser122, bothputative target sites of the inactivator DONV, are functionallyless important.     

Contribution of the C-terminal amino acid to the stability of Bacillus subtilis neutral protease

The role of the C-terminal Leu300 in maintaining thermal stabilityof the neutral protease of Bacillus subtilis was investigated.From model building studies based on the three dimensional structureof thermolysin, the neutral protease of B.thermoproteolyticus,it was conduded that this residue is located in a hydrophobicpocket composed of residues located in the C-terminal and themiddle domain. To test the hypothesis that Leu300, by contributingto a stabilizing interaction between these domains, is importantfor enzyme stability, several neutral protease mutants wereconstructed and characterized. The thermostability of the enzymewas lowered by deleting Leu300 or by replacing this residueby a smaller (Ala), a polar (Asn) or a sterically unfavourable(He) amino acid. Thermostabiity was increased upon replacingLeu300 by Phe. These results are in agreement with model-buildingstudies. The effects on thermostability observed after mutatingthe corresponding Val318 in the thermostable neutral proteaseof B.stearothermophilus were less pronounced.     

Comparative modelling of major house dust mite allergen Der p I: structure validation using an extended environmental amino acid propensity table

A model of the 3-D structure of a major house dust mite allergenDer p I associated with hypersensitivity reactions in humanswas built from its amino acid sequence and its homology to threeknown structures, papain, actinidin and papaya proteinase flof the cysteine proteinase family. Comparative modelling usingCOMPOSER was used to arrive at an initial model. This was refinedusing interactive graphics and energy minimization with theAMBER force field incorporated in SYBYL (Tripos Associates).Compatibility of the Der p I amino add sequence with the cysteineproteinase fold was checked using an environment-dependent aminoadd propensity table incorporated into a new program HARMONYwith a variable length windowing facility. A fiveresidue windowwas used to probe local conformational integrity. Propensitieswere derived from a structural alignment database of homologousproteins using a robust entropy-driven smoothing procedure.Der p I shares essential structural and mechanistic featureswith other papain-like cysteine proteinases, including cathepsinB. The active-site t iolate-imidazolium ion pair comprises theside chains of Cys34 and Hisl70. A cystine disulfide not presentin other known structures bridges residue 4 of an N-terminalextension and the core residue 117. Two conserved disulfidebridges are formed by residues 31 and 71 and residues 65 and103. Model building of peptide substrate analogue complexessuggests a preference for phenylalanyl or bask residues at theP₂ position, whilst selectivity may be of minor importance atthe S₁ subsite. The electrostatic influences on the Der p Iactive-site ion pair and extended peptide binding region aremarkedly different from those in known structures. A highlyimmunogenic surface exposed region (residues 107–131),comprising several overlapping T cell epitope sites, has noshared sequence identity with human liver cathepsin B and containsthree insertion-deletion sites. The structure provides a basisfor testing the substrate specificity of Der p I and the potentialrole of proteinase activity in hypersensitivity reactions. Thesestudies may offer a new treatment strategy by hyposensitizationwith inactive mutants or mutants with significantly alteredproteinase activity, either alone or complexed with antibody.     

Protein engineering of alcohol dehydrogenase -- 1. Effects of two amino acid changes in the active site of yeast ADH-1

One of the promises held out by protein engineering is the abilityto alter predictably the properties of an enzyme to enable itto find new substrates or catalyse existing substrates moreefficiently, such manipulations being of interest both enzymologicallyand, potentially, industrially. It has been postulated thatin yeast alcohol dehydrogenase (YADH-1) certain amino acidssuch as Trp 93 and Thr 48 constrict the active site due to theirbulky side chains and thus impede catalysis of molecules largerthan ethanol. To study effects of enlarging the active sitewe have made two changes into YADH-1, replacing Trp 93 withPhe and Thr 48 with Ser. Kinetic experiments showed that thisenzyme had marked increases in reaction velocity for the n-alcoholspropanol, butanol, pentanol, hexanol, heptanol, octanol andcinnamyl alcohol compared to the parent, agreeing with the predictionthat expanding the active site should facilitate the oxidationof larger alcohols. The substrate affinities were slightly reducedin the altered enzyme, possibly due to its having reduced hydrophobicityat Phe 93.     

Phe496 and Leu497 are essential for receptor binding and cytotoxic action of the murine interleukin-4 receptor targeted fusion toxin DAB389-mIL-4

DAB₃₈₉-mIL-4 is a murine interleukin-4 (mIL-4) diphtheria toxin-relatedfusion protein which has been shown to be selectively toxicto cells expressing the mIL-4 receptor. In this report, we haveused site-directed and in-frame deletion mutagenesis to studythe role of the putative C-terminal -helix (helix E) of themIL-4 component of DAB₃₈₉-mIL-4 in the intoxication process.We demonstrate that deletion of the C-terminal 15 amino acidsof the fusion toxin leads to loss of cytotoxicity. The substitutionof Phe496 with either Pro, Ala or Tyr, results in a > 20-folddecrease in cytotoxic activity of the respective mutant fusiontoxins. In addition, substitution of Leu497 with either Alaor Glu results in a similar loss of cytotoxic activity. Allof these mutant forms of the mIL-4 fusion toxin demonstratea significant decrease in binding affinity (K_i) to the mIL-4receptor in a competitive radioligand binding assay. In markedcontrast, however, the substitution of Asp495 with Asn resultsin a 4-fold increase in cytotoxic potency and binding affinityto mIL-4 receptor bearing cells in vitro.