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1.
Effect of amino acid deletions in the O-glycosylated region of Aspergillus awamori glucoamylase 总被引:1,自引:0,他引:1
Libby Carol Baker; Cornett Catherine A.G.; Reilly Peter J.; Ford Clark 《Protein engineering, design & selection : PEDS》1994,7(9):1109-1114
Aspergillus awamori glucoamylase (GA) contains globular catalyticand starch-binding domains (residues 1471 and 509616,respectively). A heavily O-glycosylated sequence comprises twoparts. The first (residues 441471) in the crystal structurewraps around an /-barrel formed by residues 1440. Thesecond (residues 472508) is an extended, semi-rigid linkerbetween the two domains. To investigate the functional roleof this linker, we made internal deletions to remove residues466512 (GA1), 485512 (GA2) and 466483 (GA3).GA2 and GA3 were expressed in Saccharomyces cerevisiae culturesupernatants at 60 and 20% the wild-type level, respectively,while GA1 was almost undetectable. Western blots comparing extracellularand intracellular fractions indicated that the region deletedin GA3 was critical for secretion, while the region deletedin GA2 contributed to the production of a stable enzyme structure.The activities of purified GA2 and GA3 on soluble and insolublestarch were similar to those of wild-type GA, indicating thatfor soluble starch their deletions did not affect the catalyticdomain and for insoluble starch the linker does not coordinatethe activities of the catalytic and starch-binding domains.The deletions had a significant negative effect on GA2 and GA3thermos tabilities. 相似文献
2.
Coutinho Pedro M.; Reilly Peter J. 《Protein engineering, design & selection : PEDS》1994,7(3):393-400
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 68 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. 相似文献
3.
Sierks Michael R.; Ford Clark; Reilly Peter J.; Svensson Birte 《Protein engineering, design & selection : PEDS》1989,2(8):621-625
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 (kcat) of wild-type glucoamylase towards maltose andmaltoheptaose. MichaelLs constants (KM) 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. 相似文献
4.
Identification and elimination by site-directed mutagenesis of thermolabile aspartyl bonds in Aspergillus awamori glucoamylase 总被引:2,自引:0,他引:2
Chen Hsiu-mei; Ford Clark; Reilly Peter J. 《Protein engineering, design & selection : PEDS》1995,8(6):575-582
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 Aspl26Glyl27 bond was preferentiallycleaved at pH 3.5,while at pH 4.5 cleavage of the Asp257Pro258and Asp293Gly294 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 Asp257Pro258and Asp293Gly294 bonds. The Asp257Glu mutant was producedvery slowly and was more thermostable than wildtype 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. 相似文献
5.
Flory Nathalie; Gorman Maureen; Coutinho Pedro M.; Ford Clark; Reilly Peter J. 《Protein engineering, design & selection : PEDS》1994,7(8):1005-1012
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 相似文献
6.
Sierks Michael R.; Svensson Blrte 《Protein engineering, design & selection : PEDS》1994,7(12):1479-1484
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. 相似文献
7.
Bakir Ufuk; Coutinho Pedro M.; Sullivan Patrick A.; Ford Clark; Reilly Peter J. 《Protein engineering, design & selection : PEDS》1993,6(8):939-946
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 kcat but to no changein Km, verifying this residue's catalytic function. Aspl76-Gluand Glul80-Asp mutations affected Km a more than kcat, implyingthat Aspl76 and Glul80 are involved in substrate binding orstructural integrity. The Leul77-Asp mutation decreased kcatonly moderately, probably by changing the position of the generalacid catalytic group, and did not affect Km. The Trpl78-Aspmutation greatly decreased kcat while increasing Km, 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. 相似文献
8.
Sierks Michael R.; Ford Clark; Reilly Peter J.; Svensson Birte 《Protein engineering, design & selection : PEDS》1993,6(1):75-79
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 kcat 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. KM 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 kcat or KM 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. 相似文献
9.
Jemth P Johnson CM Gianni S Fersht AR 《Protein engineering, design & selection : PEDS》2008,21(3):207-214
The role of intermediates in the folding reaction of single-domainproteins is a controversial issue. It was previously shown bydifferent methods that an on-pathway intermediate is populatedin the presence of sodium sulphate during the folding of theFF domain from HYPA/FBP11. Here we demonstrate using analysisof the amplitudes of kinetic traces that this burst-phase foldingintermediate is present at different salt concentration andat various pH, and is also found in roughly 30 site-directedmutants. The intermediate appears robust to changing conditionsand thus fulfils an important criterion for a productive molecularspecies on the folding reaction pathway. 相似文献
10.
Coutinho Pedro M.; Reilly Peter J. 《Protein engineering, design & selection : PEDS》1994,7(6):749-760
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. 相似文献
11.
Liu Hsuan-Liang; Ford Clark; Reilly Peter J. 《Protein engineering, design & selection : PEDS》1999,12(2):163-172
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. 相似文献
12.
Hughes P.E.; Handford P.A.; Austen D.E.G.; Brownlee G.G. 《Protein engineering, design & selection : PEDS》1994,7(9):1121-1127
Vitamin K-dependent plasma proteins contain a highly conservedhydrophobic domain located between the -carboxyglutamic acid(Gla) domain and the first epidermal growth factor (EGF)-likedomain. Here we have used protein engineering of the hydrophobicdomain in human factor IX to investigate its function in intactfactor IX. Mutant proteins were generated by site-directed mutagenesisand in vitro expression in Madin-Darby canine kidney (MDCK)cells. All of our mutants, including one with a deletion ofthe entire hydrophobic domain, were activated by factor XIa,showing that this domain is not required for factor IX activation.The results with the mutant Phe41 Val suggest that the hydrophobicdomain interacts with the adjacent EGF-like domain. Our datafor the Phe41 Asp mutant is consistent with, but cannot prove,a role for this residue in the maintenance of a phospholipid-bindingstructure required for factor IX function. 相似文献
13.
Ayvazian Laurence; Crenon Isabelle; Granon Simone; Chapus Catherine; Kerfelec Brigitte 《Protein engineering, design & selection : PEDS》1996,9(8):707-711
The organization of the pancreatic lipase in two well defineddomains has been correlated to a specific function for eachdomain, catalytic activity for the N-terminal domain and colipasebinding for the C-terminal domain. In order to see if such anorganization implies that the two domains can behave as separateentities, we expressed the N- and C-terminal domains in insectcells. The recombinant proteins secreted in the cell supernatantspresent the expected molecular properties. However, whereasthe C-terminal domain retains its function of colipase binding,the N-terminal domain appears to be unable to ensure catalysis.The lack of activity of the recombinant N-terminal domain couldresult either from a (partially) incorrect folding or from anincapacity to function by itself. These results suggest that,although both are structurally well defined, the two domainsof the pancreatic lipase behave differently when they are expressedas separate entities. 相似文献
14.
Prevost Martine; Kocher Jean-Pierre 《Protein engineering, design & selection : PEDS》1999,12(6):475-483
The structure and dynamics of the lipid-free LDL-receptor-bindingdomain of apolipoprotein E (apoE-RBD) has been investigatedby Molecular Dynamics Simulations. ApoE-RBD in its monomericlipid-free form is a singular four-helix bundle made up of fourelongated amphipathic helices. Analysis of one 1.5 ns moleculardynamics trajectory of apoE-RBD performed in water indicatesthat the lipid-free domain adopts a structure that exhibitscharacteristics found in native proteins: it has very stablehelices and presents a compact structure. Yet its interior exhibitsa larger number of transient atomic-size cavities relative tothat found in other proteins of similar size and its apolarside chains are more mobile. The latter features distinguishthe elongated four-helix bundle as a slightly disordered structure,which shows a structural likeness with some de novo designedfour-helix bundle proteins and shares with the latter a leucine-richresidue composition. We anticipate that these unique propertiescompared with other native helix bundles may be related to thepostulated ability of apoE-RBD to undergo an opening of itsbundle upon interaction with phospholipids. The distributionof empty cavities computed along the trajectory in the interfaceregions between the different pairs of helices reveals thatthe tertiary contacts in one of the interfaces are weaker suggestingthat this particular interface could be more easily rupturedupon lipid association. 相似文献
15.
Uegaki Koichi; Shirakawa Masahiro; Fujita Takashi; Taniguchi Tadatsugu; Kyogoku Yoshimasa 《Protein engineering, design & selection : PEDS》1993,6(2):195-200
The DNA binding domain of the interferon regulatory factor-2protein (IRF-2) has been produced and characterized, -chymotrypsindigestion of the purified IRF-2 protein bound to a syntheticbinding site yields a peptide fragment of 14 K in molecularweight. N-terminal analysis of this peptide fragment showedthat its sequence is the same as that of the intact IRF-2. Apeptide fragment of {small tilde} 14 K, IRF-2(113), which correspondsto the N-terminal 113 amino acids of the intact IRF-2 protein,has been expressed in a functional form in Escherichia coli.The first methionine was processed during the expression andthe purified IRF-2(113) thus contains 112 amino acids. DNaseI footprinting and gel retardation assaying showed that IRF-2(113)binds to a synthetic DNA having the consensus binding site andto the upstream regulatory sequence of the IFN-ß geneas intact IRF-2 does. These results showed that this peptidefragment, IRF-2(113), may be a good material for investigationof the DNA binding domain of IRF-2 and of the DNAproteininteraction. 相似文献
16.
Chen Hsiu-mei; Li Yuxing; Panda Tapobrata; Buehler Frdric U.; Ford Clark; Reilly Peter J. 《Protein engineering, design & selection : PEDS》1996,9(6):499-505
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 1.52-fold increased thermostabilities at pHs 3.55.5.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. 相似文献
17.
Peelman Frank; Vanloo Berlinda; Perez-Mendez Oscar; Decout Anne; Verschelde Jean-Luc; Labeur Christine; Vinaimont Nicole; Verhee Annick; Duverger Nicolas; Brasseur Robert; Vandekerckhove Joel; Tavernier Jan; Rosseneu Maryvonne 《Protein engineering, design & selection : PEDS》1999,12(1):71-78
Lecithin cholesterol acyltransferase (LCAT) is an interfacialenzyme active on both high-density (HDL) and low-density lipoproteins(LDL). Threading alignments of LCAT with lipases suggest thatresidues 5074 form an interfacial recognition site andthis hypothesis was tested by site-directed mutagenesis. The(5668) deletion mutant had no activity on any substrate.Substitution of W61 with F, Y, L or G suggested that an aromaticresidue is required for full enzymatic activity. The activityof the W61F and W61Y mutants was retained on HDL but decreasedon LDL, possibly owing to impaired accessibility to the LDLlipid substrate. The decreased activity of the single R52A andK53A mutants on HDL and LDL and the severer effect of the doublemutation suggested that these conserved residues contributeto the folding of the LCAT lid. The membrane-destabilizing propertiesof the LCAT 5668 helical segment were demonstrated usingthe corresponding synthetic peptide. An M65NN66M substitutiondecreased both the fusogenic properties of the peptide and theactivity of the mutant enzyme on all substrates. These resultssuggest that the putative interfacial recognition domain ofLCAT plays an important role in regulating the interaction ofthe enzyme with its organized lipoprotein substrates. 相似文献
18.
Assouline Z.; Shen H.; Kilburn D.G.; Warren R.A.J. 《Protein engineering, design & selection : PEDS》1993,6(7):787-792
A fusion protein, FXCBDCex, which comprises factor Xwith a cellulose-binding domain (CBDCex) fused to its C-terminus,was produced in BHK cells. It was purified from the culturemedium by affinity chromatography on cellulose. FXCBDCexcould be activated to FXaCBDCex with Russell viper venom.FXaCBDCex was as active as FXa against a chromogenicsubstrate and against proteins containing the IleGluGlyArgsequence hydrolysed by FXa. FXaCBDCex retained its activitywhen adsorbed to cellulose. 相似文献
19.
20.
Cols Neus; Romero-Isart Nuria; Bofill Roger; Capdevila Merce; Gonzalez-Duarte Pilar; Gonzalez-Duarte Roser; Atrian Silvia 《Protein engineering, design & selection : PEDS》1999,12(3):265-269
The ß domain of mouse metallothionein 1 (ßMT) wassynthesized in Escherichia coli cells grown in the presenceof copper or cadmium. Homogenous preparations of CußMTand CdßMT were used to characterize the correspondingin vivo-conformed metal-clusters, and to compare them with thespecies obtained in vitro by metal replacement to a canonicalZn3ßMT structure. The copper-containing ßMTclusters formed inside the cells were very stable. In contrast,the nascent ß peptide, although it showed cadmium bindingability, produced a highly unstable species, whose stoichiometrydepended upon culture conditions. The absence of ßMT proteinin E.coli protease-proficient hosts grown in cadmium-supplementedmedium pointed to drastic proteolysis of a poorly folded ßpeptide, somehow enhanced by the presence of cadmium. Possiblefunctional and evolutionary implications of the bioactivityof mammalian ßMT in the presence of monovalent and divalentmetal ions are discussed. 相似文献