Site-directed mutagenesis at the active site Trpl20 of Aspergillus awamori glucoamylase

Trpl20 of Aspergillus awamori glucoamylase has previously beenshown by chemical modification to be essential for activityand tentatively to be located near subsite 4 of the active site.To further test its role, restriction sites were inserted inthe cloned A.awamori gene around the Trpl20 coding region, andcassette mutagenesis was used to replace it with His, Leu, Pheand Tyr. All four mutants displayed 2% or less of the maximalactivity (k_cat) of wild-type glucoamylase towards maltose andmaltoheptaose. MichaelLs constants (K_M) of mutants decreased2- to 3-fold for maltose and were essentially unchanged formaltoheptaose compared with the wild type, except for a >3-fold decrease for maltoheptaose with the Trp120 – Tyrmutant. This mutant also bound isomaltose more strongly andhad more selectivity for its hydrolysis than wild-type glucoamylase.A subsite map generated from malto-oligosaecharide substrateshaving 2 – 7 D-glucosyl residues indicated that subsites1 and 2 had greater affinity for D-glucosyl residues in theTrp120 – Tyr mutant than in wild-type glucoamylase. Theseresults suggest that Trpl20 from a distant subsite is crucialfor the stabilization of the transition-state complex in subsites1 and 2.     

Identification and elimination by site-directed mutagenesis of thermolabile aspartyl bonds in Aspergillus awamori glucoamylase

Both Dative Aspergillus niger glucoamylase and wild-type Aspergillusawamori glucoamylase expressed in Saccharo-myces cerevisiae,which have identical primary structures, undergo hydrolysisat aspartyl bonds at low pH values and elevated temperatures.In native A.niger enzyme the Aspl26–Glyl27 bond was preferentiallycleaved at pH 3.5,while at pH 4.5 cleavage of the Asp257–Pro258and Asp293–Gly294 bonds was dominant. In wild-type A.awamoriglucoamylase, cleavage of the latter was dominant at both pH3.5 and 4.5. Site-directed mutations Aspl26Glu and Glyl27Alain wild-type enzyme decreased specific activities by 60 and30%, respectively, and increased irreversible thermoinactivationrates 3- to 4-fold at pH 4.5. Replacement of Asp257 with Gluand Asp293 with Glu or Gin decreased specific activities by20%, but greatly reduced cleavage of the Asp257–Pro258and Asp293–Gly294 bonds. The Asp257Glu mutant was producedvery slowly and was more thermostable than wild–type glucoamylaseat pH 4.5up to 70°C. Replacement of Asp293 with either Gluor Gln significantly raised protein production and slightlyincreased thermostability at pH 3.5 and 4.5, but not at pH5.     

Deletion analysis of the starch-binding domain of Aspergillus glucoamylase

The large form of glucoamylase (GAI) from Aspergillus awamori(EC 3.2.1.3 [EC] ) binds strongly to native granular starch, whereasa truncated form (GAII) which lacks 103 C-terminal residues,does not. This C-terminal region, conserved among fungal glucoamylasesand other starch-degrading enzymes, is part of an independentstarch-binding domain (SBD). To investigate the SBD boundariesand the function of conserved residues in two putative substrate-bindingsites, five gluco-amylase mutants were constructed with extensivedeletions in this region for expression in Saccharomyces cerevisiae.Progressive loss of both starch-binding and starch-hydrolyticactivity occurred upon removal of eight and 25 C-terminal aminoacid residues, or 21 and 52 residues close to the N-terminus,confirming the requirement for the entire region in formationof a functional SBD. C-terminal deletions strongly impairedSBD function, suggesting a more important role for one of theputative binding sites. A GAII phenocopy showed a nearly completeloss of starch-binding and starch-hydrolytic activity. The deletionsdid not affect enzyme activity on soluble starch or thermo-stabilityof the enzyme, confirming the independence of the catalyticdomain from the SBD.     

Cassette mutagenesis of Aspergillus awamori glucoamylase near its general acid residue to probe its catalytic and pH properties

Nine single amino add mutations in the active site of Aspergillusawamori glucoamylase were made by cassette mutagenesis to alterthe pH dependence of the enzyme and to determine possible functionsof the mutated residues. The Glul79-Asp mutation expressed inyeast led to a very large decrease in k_cat but to no changein K_m, verifying this residue's catalytic function. Aspl76-Gluand Glul80-Asp mutations affected K_m a more than k_cat, implyingthat Aspl76 and Glul80 are involved in substrate binding orstructural integrity. The Leul77-Asp mutation decreased k_catonly moderately, probably by changing the position of the generalacid catalytic group, and did not affect K_m. The Trpl78-Aspmutation greatly decreased k_cat while increasing K_m, showingthe importance of Trpl78 in the active site. Vall81-Asp andAsnl82-Asp mutations changed kinetk values little, suggestingthat Vall81 and Asnl82 are of minor catalytic and structuralimportance. Finally, insertions of Asp or Gly between residues176 and 177 resulted in almost complete loss of activity, probablycaused by destruction of the active site structure. No largechanges in pH dependence occurred in those mutations where kineticvalues could be determined, in spite of the increase in mostcases of the total negative charge. Increases in activationenergy of maltoheptaose hydrolysis in most of the mutant glucoamylasessuggested cleavage of individual hydrogen bonds in enzyme-substratecomplexes.     

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     

Protein engineering of the relative specificity of glucoamylase from Aspergillus awamori based on sequence similarities between starch-degrading enzymes

Aspergillus glucoamylase catalyzes hydrolysis of D-glucose fromnon-reducing ends of starch with an {small tilde}300-fold {k^JKm) preference for the a-1,4- over the a-l,6-glucosidic linkagedetermined using the substrates maltose and iso-maltose. Itis postulated that as most amylolytic enzymes act on eitherthe a-1,4- or a-l,6-linkages, sequence comparison between active-siteregions should enable the correlation of the substrate bondspecificity with particular residues at key positions. Therefore,the already high bond-type selectivity in Aspergillus glucoamylasecould theoretically be augmented further by three single mutations,Serll9 Tyr, Glyl83 Lys and Serl84 His, in two separate active-siteregions. These mutants all had slight increases in activityas compared with the wild-type enzyme towards the a-l,4-linkedmaltose; this was due to lower Km values as well as small decreasesin activity towards isomaltose. This latter decrease in activitywas a result of higher Km values and a decrease in fc^, forthe Serl84 His mutant As a consequence, the selectivity of thethree glucoamylase mutants for a-1,4- over a-l,6-linked disaccharidesis enhanced 2.3- to 3.5-fold. In addition, the introductionof a cationic side chain in Glyl83 Lys and Serl84 His glucoamylase,broadens the optimal pH range for activity towards acidic aswell as alkaline conditions.     

Functional roles and subsite locations of Leu177, Trp178 and Asn182 of Aspergillus awamori glucoamylase determined by site-directed mutagenesis

Fungal glucoamylases contain four conserved regions. One regionfrom the Aspergillus niger enzyme contains three key carboxylicacid residues, the general acid catalytic group, Glu179, alongwith Asp176 and Glu180. Three site-directed mutations, Leu177– His, Trp178 – Arg and Asn182 – Ala, wereconstructed near these acidic groups to reveal the functionof other conserved residues in this region. Leu177 and Trp178are strictly conserved among fungal glucoamylases, while anamide, predominantly Asn, always occurs at position 182. Substitutionsof Leu177 or Trp178 cause significant decreases in k_cat withthe substrates tested. Similar increases in activation energiesobtained with Leu177 – His with both -(1,4)- and -(1,6)-linkedsubstrates indicate Leu177 is located in subsite 1. K_M valuesobtained with the Trp178 – Arg mutation increase for an-(1,6)-linked substrate, but not for -(1,4)-linked substrates.Calculated differences in activation energy between substratesindicate Trp178 interacts specifically with subsite 2. The Asn182 Ala mutation did not change k_cat or K_M values, indicating thatAsn182 is not crucial for activity. These results support amechanism for glucoamylase catalytic activity consisting ofa fast substrate binding step followed by a conformational changeat subsite 1 to stabilize the transition state complex.     

Structure-function relationships in the catalytic and starch binding domains of glucoamylase

Sixteen primary sequences from five sub-families of fungal,yeast and bacterial glucoamylases were related to structuralinformation from the model of the catalytic domain of Aspergillusawamori var. X100 glucoamylase obtained by protein crystallography.This domain is composed of thirteen -belices, with five conservedregions defining the active site. Interactions between methyl-maltoside and active site residues were modelled, and the importanceof these residues on the catalytic action of different glucoamylaseswas shown by their presence in each primary sequence. The overallstructure of the starch binding domain of some fungal glucoamylaseswas determined based on homology to the Cterminal domains ofBacillus cyclodextrin glucosyltransferases. Crystallographyindicated that this domain contains 6–8 ß-strandsand homology allowed the attribution of a disulfide bridge inthe glucoamylase starch binding domain. Glucoamylase residuesThr525, Asn530 and Trp560, homologous to Bacillus stearothermophiluscyclodextrin glucosyltransferase residues binding to maltosein the Cterminal domain, could be involved in raw-starch binding.The structure and length of the linker region between the catalyticand starch binding domains in fungal glucoamylases can varysubstantially, a further indication of the functional independenceof the two domains.     

Effect of replacing helical glycine residues with alanines on reversible and irreversible stability and production of Aspergillus awamori glucoamylase

To decrease irreversible thermoinactivation of Aspergillus awamoriglucoamylase, five Gly residues causing helix flexibility werereplaced with Ala residues. Mutation of Gly57 did not affectthermostability. Mutation of Gly137 doubled it at pHs 3.5 and4.5 but barely changed it at pH 5.5. The Gly139Ala mutationdid not change thermostability at pH 3.5, improved it at pH4.5 and worsened it at pH 5.5. The Gly137/Gly139Ala/Ala mutationgave 1.5–2-fold increased thermostabilities at pHs 3.5–5.5.Mutations of Gly251 and Gly383 decreased it at all pHs. Gly137Alaand Gly137/Gly139Ala/Ala glucoamylases are the most stable yetproduced by mutation. Guanidine treatment at pH 4.5 decreasedthe reversible stabilities of Gly137Ala, Gly139Ala and Gly137/Gly139Ala/Alaglucoamylases at infinite dilution while not changing thoseof Gly251Ala and Gly 383Ala glucoamylases, which is, in general,opposite to what occurred with thermoinactivation. Mutationof Gly57 greatly improved the extracellular glucoamylase productionby yeast, that of Gly137 barely affected it and those of Gly139and of both Gly137 and Gly139 strongly impeded it. These observationssuggest that -helix rigidity can affect reversible and irreversibleglucoamylase stability differently, that the effects of multiplemutations within one -helix to improve stability are not alwaysadditive and that even single mutations can strongly affectextracellular enzyme production.     

Mutations to alter Aspergillus awamori glucoamylase selectivity. IV. Combinations of Asn20->Cys/Ala27->Cys, Ser30->Pro, Gly137->Ala, 311314 Loop, Ser411->Ala and Ser436->Pro

Six previously constructed and nine newly constructed Aspergillusawamori glucoamylases with multiple mutations made by combiningexisting single mutations were tested for their ability to produceglucose from maltodextrins. Multiple mutations have cumulativeeffects on glucose yield, specific activity and thermostability.No general correlation between glucose yield and thermostabilitywas observed, although mutations that presumably impede unfoldingat high temperatures uniformly increase thermostability andgenerally increase glucose yield. Peak glucose yields decreasewith increasing temperature. The best combination of high glucoseyield, high specific activity and high thermostability occursin Asn20Cys/Ala27Cys/Ser30Pro/Gly137Ala glucoamylase.     

Structural similarities in glucoamylases by hydrophobic cluster analysis

The model of the catalytic domain of Aspergillus awamori var.X100 glucoamylase was related to 14 other glucoamylase proteinsequences belonging to five subfamilies. Structural featuresof the different sequences were revealed by multisequence alignmentfollowing hydrophobic cluster analysis. The alignment agreedwith the hydrophobic microdomains, normally conserved throughoutevolution, evaluated from the 3-D model. Saccharomyces and Clostridiumglucoamylases lack the -helix exterior to the catalytic domain.A different catalytic base was found in the Saccharomyces glucoamylasesubfamily. The starch binding domain of fungal glucoamylaseshas identical structural features and substrate interactingresidues as the C-terminal domain of models of Bacillus circulanscyclodextrin glucosyltransferases. Three putative N-glycosylationsites were found in the same turns in glucoamylases of differentsubfamilies. O-Glycosylation is present at different levelsin the catalytic domain and in the linker between the catalyticand starch binding domains.     

Altering the association properties of insulin by amino acid replacement

The importance of Pro^B28 and Lys^B29 on the self-associationof insulin was established by systematically truncating theC terminus of the B chain. The relationship between structureand association was further explored by making numerous aminoacid replacements at B²⁸ and B²⁹ Association was studied bycircular dichroism, size-exclusion chromatography and ultracentrifugation.Our results show that the location of a prolyl residue at B²⁸is critical for high-affinity self-association. Removal of Pro^B28in a series of C-terminal truncated insulins, or amino acidreplacement of Pro²⁸, greatly reduced association. The largestdisruption to association was achieved by replacing Lys^B29 withPro and varying the amino acid at B²⁸ Several of the analogswere predominantly monomers in solutions up to 3 mg/ml. Theseamino acid substitutions decreased association by primarilydisrupting the formation of dimers. Such amino acid substitutionsalso substantially reduced the Zn-induced insulin hexamer formation.The formation of monomeric insulins through amino acid replacementswas accompanied by conformational changes that may be the causefor decreased association. It is demonstrated that self-associationof insulin can be drastically altered by substitution of oneor two key amino acids.     

Coordinated amino acid changes in homologous protein families

In the tobamovirus coat protein family, amino acid residuesat some spatially close positions are found to be substitutedin a coordinated manner [Altschuh et al. (1987) J. Mol. Biol.,193,693]. Therefore, these positions show an identical patternof amino acid substitutions when amino acid sequences of thesehomologous proteins are aligned. Based on this principle, coordinatedsubstitutions have been searched for in three additional proteinfamilies: serine proteases, cysteine proteases and the haemoglobins.Coordinated changes have been found in all three protein familiesmostly within structurally constrained regions. This methodworks with a varying degree of success depending on the functionof the proteins, the range of sequence similarities and thenumber of sequences considered. By relaxing the criteria forresidue selection, the method was adapted to cover a broaderrange of protein families and to study regions of the proteinshaving weaker structural constraints. The information derivedby these methods provides a general guide for engineering ofa large variety of proteins to analyse structure–functionrelationships.     

Concurrent mutations in six amino acids in beta-glucuronidase improve its thermostability

To achieve a thermostable beta-glucuronidase (GUS) and identify key mutation sites, we applied in vitro directed evolution strategy through DNA shuffling and obtained a highly thermostable mutant GUS gene, gus-tr, after four rounds of DNA shuffling and screening. This variant had mutations in 15 nucleic acid sites, resulting in changes in 12 amino acids (AAs). Using gus-tr as the template, we further performed site-directed mutagenesis to reverse the individual mutation to the wild-type protein. We found that six sites (Q493R, T509A, M532T, N550S, G559S and N566S) present in GUS-TR3337, were the key AAs needed to confer its high thermostability. Of these, Q493R and T509A were not reported previously as important residues for thermostability of GUS. Furthermore, all of these six mutations must be present concurrently to confer the high thermostability. We expressed the gus-tr3337 gene and purified the GUS-TR3337 protein that contained the six AA mutations. Compared with the wild-type protein which lost its activity completely after 10 min at 70 degrees C, the mutant GUS-TR3337 protein retained 75% of its activity when heated at 80 degrees C for 10 min. The GUS-TR3337 exhibited high activity even heated at 100 degrees C for 30 min on nitrocellulose filter. The comparison of molecular models of the mutated and wild-type enzyme revealed the relation of protein function and these structural modifications.     

Effect of linker sequences between the antibody variable domains on the formation, stability and biological activity of a bispecific tandem diabody

Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (V(H) and V(L)) domains of two different specificities connected by three linkers. When assembled in the order V(H)(A)-linker(1)-V(L)(B)-linker(2)-V(H)(B)-linker(3)-V(L)(A), the single-chain molecule either folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody, or forms a homodimer that is twice as large, a so-called tandem diabody. The formation of the tandem diabody is determined by the association of complementary V(H) and V(L) domains located on different polypeptide chains, and depends on the length and probably the amino acid composition of the three linkers joining the variable domains. We generated a number of single-chain constructs using four V(H) and V(L) domains specific either for human CD3, a component of T-cell receptor (TCR) complex, or for CD19, a human B-cell antigen, separated by different rationally designed peptide linkers of 6-27 amino acid residues. The generated bispecific constructs were expressed in bacterial periplasm and their molecular forms, antigen-binding properties, stability, and T-cell proliferative and anti-tumor activities were compared. Using peripheral blood mononuclear cell cultures from patients suffering from B-cell chronic lymphocytic leukemia, we demonstrated that the tandab-mediated activation of autologous T cells and depletion of malignant cells correlates with the stability of the recombinant molecule and with the distance between the CD19 and CD3 binding sites.     

Replacement and deletion mutations in the catalytic domain and belt region of Aspergillus awamori glucoamylase to enhance thermostability

Three single-residue mutations, Asp71     

Site-directed mutagenesis study on the roles of evolutionally conserved aspartic acid residues in human glutathione S-transferase P1-1

The evolutionally conserved aspartyl residues (Asp57, Asp98and Asp152) in human glutathione S-transferase P1-1 were replacedwith alanine by site-directed mutagenesis to obtain the mutants(D57A, D98A and D152A). The replacement of Asp98 with alanineresulted in a decrease of the affinity for S-hexyl-GSH-agarose,a 5.5-fold increase of the K_m^GHS and a 2.9-fold increase ofthe I₅₀ of S-hexyl-GSH for GSH–CDNB conjugation. Asp98seems to participate in the binding of GSH through hydrogenbonding with the -carboxylate of the -glutamyl residue of GSH.The k_cat of D98A was 2.6-fold smaller than that of the wild-type,and the pK_a of the thiol group of GSH bound in D98A was {smalltilde}0.8 pK units higher than those in the wild-type. Asp98also seems to contribute to the activation of GSH to some extent.On the other hand, most of the kinetic parameters of D57A andD152A were similar to those of the wild-type. However, the thermostabilitiesof D57A and D152A were significantly lower than that of thewild-type. Asp57 and Asp152 seem to be important for maintainingthe proper conformation of the enzyme.     

Effect on thermostability and catalytic activity of introducing disulfide bonds into Aspergillus awamori glucoamylase

Two additional disulfide bonds and three combined thermostabilizing mutations were introduced into Aspergillus awamori glucoamylase to test their effects on enzyme thermostability and catalytic properties. The single cysteine mutations N20C, A27C, T72C and A471C were made and combined to produce the double cysteine mutations N20C/ A27C and T72C/A471C. The double cysteine mutants were expressed efficiently in Saccharomyces cerevisiae, and disulfide bonds formed spontaneously after fermentation. At 50 degrees C, the single mutants N20C and A27C had decreased specific activity, whereas the specific activity of the double mutants N20C/A27C and T72C/A471C were similar to wild-type glucoamylase. The N20C/A27C mutation increased thermostability, with an increased activation free energy of 1.5 kJ/mol at 65 degrees C, while the single mutation A27C only slightly increased thermostability and N20C decreased it. The other disulfide bond-forming mutation T72C/A471C did not affect thermostability at pH 4.5. The N20C/A27C mutation was separately combined with two other thermostabilizing mutations, G137A and S436P. Thermostabilities of all of the combined mutated glucoamylases were additive. N20C/A27C/G137A glucoamylase had higher specific activity than wild-type glucoamylase from 45 to 67.5 degrees C. The disulfide bond between positions 20 and 27 connects the C- terminus of helix 1 and the following beta-turn, suggesting that this region is important for glucoamylase thermostability.      

Effects of signal peptide changes on the secretion of bovine somatotropin (bST) from Escherichia coli

Bovine somatotropin (bST) was secreted from Escherichia coliat moderate levels of 1–2 µg/ml/OD using expressionvectors in which the bST gene was fused to the lamB secretionsignal. To study the secretion properties of bST in E.coli further,two approaches for modifying the secretion signal were employed.In the first case, fusion proteins were constructed with sixalternative bacterial secretion signals: three from E.coli proteins(HisJ, MalE and OmpA), two from bacteriophage proteins (M13coat protein and PA-2 Lc) and one from the chitinase A proteinof Serratia marcescens. The results, as monitored by Westernblot analysis of both total cell protein and the periplasmicfraction, showed that these changes in the secretion signaldid not significantly affect the secretion properties of bST.In the second approach, a library of random mutations was createdin the lamB secretion signal and 200 independent clones werescreened. The level of secreted bST was determined by growingindividual clones in duplicate in microtiter wells, inducingprotein expression and measuring the bST released by osmoticshock using a particle concentration fluorescent immunoassay.The secretion properties of several novel variants in the LamBsignal peptide are presented.     

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.