Improvement of Oxidative and Thermostability of N-Carbamyl-D-Amino Acid Amidohydrolase by Directed Evolution

N-Carbamyl-D-amino acid amidohydrolase (N-carbamoylase), whichis currently employed in the industrial production of unnaturalD-amino acid in conjunction with D-hydantoinase, has low oxidativeand thermostability. We attempted the simultaneous improvementof the oxidative and thermostability of N-carbamoylase fromAgrobacterium tumefaciens NRRL B11291 by directed evolutionusing DNA shuffling. In a second generation of evolution, thebest mutant 2S3 with improved oxidative and thermostabilitywas selected, purified and characterized. The temperature atwhich 50% of the initial activity remains after incubation for30 min was 73°C for 2S3, whereas it was 61°C for wild-typeenzyme. Treatment of wild-type enzyme with 0.2 mM hydrogen peroxidefor 30 min at 25°C resulted in a complete loss of activity,but 2S3 retained about 79% of the initial activity under thesame conditions. The K_m value of 2S3 was estimated to be similarto that of wild-type enzyme; however k_cat was decreased, leadingto a slightly reduced value of k_cat/K_m, compared with wild-typeenzyme. DNA sequence analysis revealed that six amino acid residueswere changed in 2S3 and substitutions included Q23L, V40A, H58Y,G75S, M184L and T262A. The stabilizing effects of each aminoacid residue were investigated by incorporating mutations individuallyinto wild-type enzyme. Q23L, H58Y, M184L and T262A were foundto enhance both oxidative and thermostability of the enzymeand of them, T262A showed the most significant effect. V40Aand G75S gave rise to an increase only in oxidative stability.The positions of the mutated amino acid residues were identifiedin the structure of N-carbamoylase from Agrobacterium sp. KNK712 and structural analysis of the stabilizing effects of eachamino acid substitution was also carried out.     

Expression of deletion constructs of bovine {beta}-1, 4-galactosyltransferase in Escherichia coli: importance of Cysl34 for its activity

Bovine ß-1, 4-galactosyltransferase (ß-1,4-GT; EC 2.4.1.90 [EC] ) belongs to the glycosyltransferase familyand as such shares a general topology: an N-terminal cytoplasmictail, a signal anchor followed by a stem region and a catalyticdomain at the C-tenninal end of the protein. cDNA constructsof the N-terminal deleted forms of ß-1, 4-GT wereprepared in pGEX-2T vector and expressed in E.coli as glutathione-S-transferase(GST) fusion proteins. Recombinant proteins accumulated withininclusion bodies as insoluble aggregates that were solubilizedin 5 M guanidine HCl and required an ‘oxido-shuffling’reagent for regeneration of the enzyme activity. The recombinant(ß-1, 4-GT, devoid of the GST domain, has 30–85%of the sp. act. of bovine milk ß-1, 4-GT with apparentK_ms for N-acetylglucosamine and UDP-galactose similar to thoseof milk enzyme. Deletion analysesshow that both (ß-1,4-GT and lactose synthetase activities remain intact even inthe absence of the first 129 residues (pGT-dl29). The activitiesare lost when either deletions extend up to residue 142 (pGT-dl42)or Cysl34 is mutatedto Ser (pGT-dl29C134S). These results suggestthat the formation of a disulfide bond involving Cysl34 holdsthe protein in a conformation that is required for enzymaticactivity.     

Contribution of a disulfide bridge to the stability of 1,3-1,4-P-D-glucan 4-glucanohydrolase from Bacillus licheniformis

Bacillus 1,3-1,4-ß-glucanases possess a highly conserveddisulfide bridge connecting a ß-strand with a solventexposedloop lying on top of the extended binding site cleft The contributionof the disulfide bond and of both individual cysteines (Cys61and Cys90) in the Bacillus licheniformis enzyme to stabilityand activity has been evaluated by protein engineering methods.Reduction of the disulfide bond has no effect on kinetic parameters,has only a minor effect on the activity-temperature profileat high temperatures, and destabilizes the protein by less than0.7 kcal/mol as measured by equilibrium urea denatu ration at37°C. Replacing either of the Cys residues with Ala destabilizesthe protein and lowers the specific activity. C90A retains 70%of wild-type (wt) activity (in terms of Vmax), whereas C61Aand the double mutant C61A–C90A have 10% of wt V_max. Alarger change in free energy of unfolding is seen by equilibriumurea denaturation for the C61A mutation (loop residue, 3.2 kcal/molrelative to reduced wt) as compared with the C90A mutation (ß-strandresidue, 1.8 kcal/mol relative to reduced wt), while the doublemutant C61A–C90A is 0.8 kcal/mol less stable than thesingle C61A mutant. The effects on stability are interpretedas a result of the change in hydrophobic packing that occursupon removal of the sulfur atoms in the Cys to Ala mutations     

The sequence and X-ray structure of the trypsin from Fusarium oxysporum

The trypsin from Fusarium oxysporum is equally homologous totrypsins from Streptomyces griseus, Streptomyces erythraeusand to bovine trypsin. A DFP (diisopropylfluorophosphate) inhibitedform of the enzyme has been crystallized from 1.4 M Na₂SO₄,buffered with citrate at pH 5.0–5.5. The crystals belongto space group P2₁ with cell parameters a=33.43 Å, b=67.65Å, c=39.85 Å and ß=107.6°. There isone protein molecule in the asymmetric unit. X-ray diffractiondata to a resolution of 1.8 Å were collected on film usingsynchrotron radiation. The structure was solved by molecularreplacement using models of bovine and S.griseus trypsins andrefined to an R-factor of 0.141. The overall fold is similarto other trypsins, with some insertions and deletions. Thereis no evidence of the divalent cation binding sites seen inother trypsins. The covalently bound inhibitor molecule is clearlyvisible.     

Adaptation of a thermophilic enzyme, 3-isopropylmalate dehydrogenase, to low temperatures

Random mutagenesis coupled with screening of the active enzymeat a low temperature was applied to isolate cold-adapted mutantsof a thermophilic enzyme. Four mutant enzymes with enhancedspecific activities (up to 4.1-fold at 40°C) at a moderatetemperature were isolated from randomly mutated Thermus thermophilus3-isopropylmalate dehydrogenase. Kinetic analysis revealed twotypes of cold-adapted mutants, i.e. k_cat-improved and K_m-improvedtypes. The k_cat-improved mutants showed less temperature-dependentcatalytic properties, resulting in improvement of k_cat (up to7.5-fold at 40°C) at lower temperatures with increased K_mvalues mainly for NAD. The K_m-improved enzyme showed higheraffinities toward the substrate and the coenzyme without significantchange in k_cat at the temperatures investigated (30–70°C).In k_cat-improved mutants, replacement of a residue was foundnear the binding pocket for the adenine portion of NAD. Twoof the mutants retained thermal stability indistinguishablefrom the wild-type enzyme. Extreme thermal stability of thethermophilic enzyme is not necessarily decreased to improvethe catalytic function at lower temperatures. The present strategyprovides a powerful tool for obtaining active mutant enzymesat lower temperatures. The results also indicate that it ispossible to obtain cold-adapted mutant enzymes with high thermalstability.     

Module shuffling of a family F/10 xylanase: replacement of modules M4 and M5 of the FXYN of Streptomyces olivaceoviridis E-86 with those of the Cex of Cellulomonas fimi

To facilitate an understanding of structure–function relationships,chimeric xylanases were constructed by module shuffling betweenthe catalytic domains of the FXYN from Streptomyces olivaceoviridisE-86 and the Cex from Cellulomonas fimi. In the family F/10xylanases, the modules M4 and M5 relate to substrate bindingso that modules M4 and M5 of the FXYN were replaced with thoseof the Cex and the chimeric enzymes denoted FCF-C4, FCF-C5 andFCF-C4,5 were constructed. The k_cat value of FCF-C5 for p-nitrophenyl-ß-D-cellobiosidewas similar to that of the FXYN (2.2 s^–1); however, thek_cat value of FCF-C4 for p-nitrophenyl-ß-D-cellobiosidewas significantly higher (7.0 s^–1). The loss of the hydrogenbond between E46 and S22 or the presence of the I49W mutationwould be expected to change the position of Q88, which playsa pivotal role in discriminating between glucose and xylose,resulting in the increased k_cat value observed for FCF-C4 actingon p-nitrophenyl-ß-D-cellobioside since module M4directly interacts with Q88. To investigate the synergisticeffects of the different modules, module M10 of the FCF-C4 chimerawas replaced with that of the Cex. The effects of replacementof module M4 and M10 were almost additive with regard to theK_m and k_cat values.     

Contribution of engineered electrostatic interactions to the stability of cytosolic malate dehydrogenase

Protein engineering is a promising tool to obtain stable proteins.Comparison between homologous thermophilic and mesophilic enzymesfrom a given structural family can reveal structural featuresresponsible for the enhanced stability of thermophilic proteins.Structures from pig heart cytosolic and Thermus flavus malatedehydrogenases (cMDH, Tf MDH), two proteins showing a 55% sequencehomology, were compared with the aim of increasing cMDH stabilityusing features from the Thermus flavus enzyme. Three potentialsalt bridges from Tf MDH were selected on the basis of theirlocation in the protein (surface R176-D200, inter-subunit E57–K168and intrasubunit R149–E275) and implemented on cMDH usingsite-directed mutagenesis. Mutants containing E275 were notproduced in any detectable amount, which shows that the energypenalty of introducing a charge imbalance in a region that wasnot exposed to solvent was too unfavourable to allow properfolding of the protein. The salt bridge R149–E275, ifformed, would not enhance stability enough to overcome thiseffect. The remaining mutants were expressed and active andno differences from wild-type other than stability were found.Of the mutants assayed, Q57E/L168K led to a stability increaseof 0.4 kcal/mol, as determined by either guanidinium chloridedenaturalization or thermal inactivation experiments. This resultsin a 15°C shift in the optimal temperature, thus confirmingthat the inter-subunit salt bridge initially present in theT.flavus enzyme was formed in the cMDH structure and that theextra energy obtained is transformed into an increase in proteinstability. These results indicate that the use of structuralfeatures of thermophilic enzymes, revealed by a detailed comparisonof three-dimensional structures, is a valid strategy to improvethe stability of mesophilic malate dehydrogenases.     

Engineering the active center of the 6-phospho-{beta}-galactosidase from Lactococcus lactis

Several amino acids in the active center of the 6-phospho-ß-galactosidasefrom Lactococcus lactis were replaced by the corresponding residuesin homologous enzymes of glycosidase family 1 with differentspecificities. Three mutants, W429A, K435V/Y437F and S428D/K435V/Y437F, were constructed. W429A was found to have an improvedspecificity for glucosides compared with the wild-type, consistentwith the theory that the amino acid at this position is relevantfor the distinction between galactosides and glucosides. Thek_cat/K_m for o-nitrophenyl-ß-D-glucose-6-phosphate is 8-foldhigher than for o-nitrophenyl-ß-D-galactose-6-phosphatewhich is the preferred substrate of the wild-type enzyme. Thissuggests that new hydrogen bonds are formed in the mutant betweenthe active site residues, presumably Gln19 or Trp421 and theC-4 hydroxyl group. The two other mutants with the exchangesin the phosphate-binding loop were tested for their abilityto bind phosphorylated substrates. The triple mutant is inactive.The double mutant has a dramatically decreased ability to bindo-nitrophenyl-ß-D-galactose-6-phosphate whereas the interactionwith o-nitrophenyl-ß-D-galactose is barely altered. Thisresult shows that the 6-phospho-ß-galactosidase and therelated cyanogenic ß-glucosidase from Trifolium repenshave different recognition mechanisms for substrates althoughthe structures of the active sites are highly conserved.     

Cytidine deaminase from two extremophilic bacteria: cloning, expression and comparison of their structural stability

We cloned, purified and characterized two extremophilic cytidinedeaminases: CDA_Bcald and CDA_Bpsy, isolated from Bacillus caldolyticus(growth at 72°C) and Bacillus psychrophilus (growth at 10°C),respectively. We compared their thermostability also with themesophilic counterpart, CDA_Bsubt, isolated from Bacillus subtilis(growth at 37°C). The DNA fragments encoding CDA_Bcald andCDA_Bpsy were sequenced and the deduced amino acid sequencesshowed 70% identity. High sequence similarity was also foundwith the mesophilic CDA_Bsubt. Both enzymes were found to behomotetramers of approximately 58 kDa. CDA_Bcald was found tobe highly thermostable, as expected, up to 65°C, whereasCDA_Bpsy showed higher specific activity at lower temperaturesand was considerably less thermostable than CDA_Bcald. Afterpartial denaturation at 72°C for 30 min, followed by renaturationon ice, CDA_Bcald recovered 100% of its enzymatic activity, whereasCDA_Bpsy as well as CDA_Bsubt were irreversibly inactivated. Circulardichroism (CD) spectra of CDA_Bcald and CDA_Bpsy at temperaturesranging from 10 to 95°C showed a markedly different thermostabilityof their secondary structures: at 10 and 25°C the CD spectrawere indistinguishable, suggesting a similar overall structure,but as temperature increases up to 50–70°C, the -helicesof CDA_Bpsy unfolded almost completely, whereas its ß-structureand the aromatic amino acids core remained pretty stable. Nosignificant differences were seen in the secondary structuresof CDA_Bcald with increase in temperature.     

Heat labile ribonuclease HI from a psychrotrophic bacterium: gene cloning, characterization and site-directed mutagenesis

The rnhA gene encoding RNase HI from a psychrotrophic bacterium,Shewanella sp. SIB1, was cloned, sequenced and overexpressedin an rnh mutant strain of Escherichia coli. SIB1 RNase HI iscomposed of 157 amino acid residues and shows 63% amino acidsequence identity to E.coli RNase HI. Upon induction, the recombinantprotein accumulated in the cells in an insoluble form. Thisprotein was solubilized and purified in the presence of 7 Murea and refolded by removing urea. Determination of the enzymaticactivity using M13 DNA–RNA hybrid as a substrate revealedthat the enzymatic properties of SIB1 RNase HI, such as divalentcation requirement, pH optimum and cleavage mode of a substrate,are similar to those of E.coli RNase HI. However, SIB1 RNaseHI was much less stable than E.coli RNase HI and the temperature(T_1/2) at which the enzyme loses half of its activity upon incubationfor 10 min was ~25°C for SIB1 RNase HI and ~60°C forE.coli RNase HI. The optimum temperature for the SIB1 RNaseHI activity was also shifted downward by 20°C compared withthat of E.coli RNase HI. Nevertheless, SIB1 RNase HI was lessactive than E.coli RNase HI even at low temperatures. The specificactivity determined at 10°C was 0.29 units/mg for SIB1 RNaseHI and 1.3 units/mg for E.coli RNase HI. Site-directed mutagenesisstudies suggest that the amino acid substitution in the middleof the     

Directed evolution converts subtilisin E into a functional equivalent of thermitase

We used directed evolution to convert Bacillus subtilis subtilisinE into an enzyme functionally equivalent to its thermophilichomolog thermitase from Thermoactinomyces vulgaris. Five generationsof random mutagenesis, recombination and screening created subtilisinE 5-3H5, whose half-life at 83°C (3.5 min) and temperatureoptimum for activity (T_opt, 76°C) are identical with thoseof thermitase. The T_opt of the evolved enzyme is 17°C higherand its half-life at 65°C is >200 times that of wild-typesubtilisin E. In addition, 5-3H5 is more active towards thehydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide than wild-typeat all temperatures from 10 to 90°C. Thermitase differsfrom subtilisin E at 157 amino acid positions. However, onlyeight amino acid substitutions were sufficient to convert subtilisinE into an enzyme equally thermostable. The eight substitutions,which include known stabilizing mutations (N218S, N76D) andalso several not previously reported, are distributed over thesurface of the enzyme. Only two (N218S, N181D) are found inthermitase. Directed evolution provides a powerful tool to unveilmechanisms of thermal adaptation and is an effective and efficientapproach to increasing thermostability without compromisingenzyme activity.     

Alteration of aspartate 101 in the active site of Escherichia coli alkaline phosphatase enhances the catalytic activity

The function of aspartic acid residue 101 in the active siteof Escherichia coli alkaline phosphatase was investigated bysite-specific mutagenesis. A mutant version of alkaline phosphatasewas constructed with alanine in place of aspartic acid at position101. When kinetic measurements are carried out in the presenceof a phosphate acceptor, 1.0 M Tris, pH 8.0, both the k_cat andthe K_m, for the mutant enzyme increase by –2-fold, resultingin almost no change in the k_cat/K_m ratio. Under conditions ofno external phosphate acceptor and pH 8.0, both the k_cat andthe K_m for the mutant enzyme decrease by {small tilde}2-fold,again resulting in almost no change in the k_cat/K_m ratio. Thek_cat for the hydrolysis of 4-methyl-umbelliferyl phosphate andp-nitrophenyl phosphate are nearly identical for both the wild-typeand mutant enzymes, as is the K₁ for inorganic phosphate. Thereplacement of aspartic acid 101 by alanine does have a significanteffect on the activity of the enzyme as a function of pH, especiallyin the presence of a phosphate acceptor. At pH 9.4 the mutantenzyme exhibits 3-fold higher activity than the wild-type. Themutant enzyme also exhibits a substantial decrease in thermalstability: it is half inactivated by treatment at 49°C for15 min compared to 71°C for the wild-type enzyme. The datareported here suggest that this amino acid substitution altersthe rates of steps after the formation of the phospho-enzymeintermediate. Analysis of the X-ray structure of the wild-typeenzyme indicates that the increase in catalytic rate of themutant enzyme in the presence of a phosphate acceptor may bedue to an increase in accessibility of the active site nearSerl02. The increased catalytic rate of this mutant enzyme maybe utilized to improve diagnostic tests that require alkalinephosphatase, and the reduced heat stability of the mutant enzymemay make it useful in recombinant DNA techniques that requirethe ability to heat-inactivate the enzyme after use.     

Estimating the twist of {beta}-strands embedded within a regular parallel {beta}-barrel structure

The parallel ß-barrel is a recurrent structural motiffound in a large variety of different enzymes belonging to thefamily of /ß-proteins. It has been shown previouslythat the hyperboloid can be considered as a scaffold describingthe parallel ß-barrel structure. To assess restraintson ß-strand twist imposed by a given scaffold geometry,the notion of scaffold twist, T_s, is introduced. From T_s, theß-strand twist (Tw_ß) expected for a givenscaffold geometry can be derived and it is verified that thiscomputed twist can be used to identify ß-barrels characterizedby good hydrogen bonding. It is noted that Tw_ß isonly slightly affected for ß-barrels differing inthe number (N) of ß-strands, suggesting that restraintson main-chain conformation of ß-strands are not likelyto account for the N = 8 invariability observed in natural parallelß-barrels thereby strengthening previous work rationalizingthis constancy.     

Breakdown in the relationship between thermal and thermodynamic stability in an interleukin-1{beta} point mutant modified in a surface loop

Sequence variants of the ß-barrel protein interleukin-1ßhave been analyzed for their stabilities toward irreversiblethermal inactivation by monitoring the generation of light scatteringaggregates on heating. The derived temperatures for the onsetof aggregation (T_agg values) correlate well with the free energiesof unfolding of these proteins with the exception of one variant,Lys97—Val (K97V), which undergoes aggregation at a temperature7°C lower than expected based on its thermodynamic stability.This lower than expected thermal stability may be due to generationof an aggregation-prone unfolding intermediate at a temperaturelower than the T_m of the global transition. This hypothesisis supported by the location of residue 97 in the long 86–99loop which has structural features suggesting it may comprisea small, independent folding unit or microdomain. The excellentcorrelation of thermal and thermodynamic stabilities of sevenof the eight variants tested is consistent with accepted modelsfor thermal inactivation of proteins. At the same time the poorfit of the K97V variant underscores the risk in using thermalstability data in quantitative analysis of mutational studiesof the folding stability of proteins.     

Thermal unfolding and conformational stability of the recombinant domain II of glutamate dehydrogenase from the hyperthermophile Thermotoga maritima

Domain II (residues 189–338, M_r = 16 222) of glutamatedehydrogenase from the hyperthermophilic bacterium Thermotogamaritima was used as a model system to study reversible unfoldingthermodynamics of this hyperthermostable enzyme. The proteinwas produced in large quantities in E.coli using a T7 expressionsystem. It was shown that the recombinant domain is monomericin solution and that it comprises secondary structural elementssimilar to those observed in the crystal structure of the hexamericenzyme.The recombinant domain is thermostable and undergoesreversible and cooperative thermal unfolding in the pH range5.90–8.00 with melting temperatures between 75.1 and 68.0°C.Thermal unfolding of the protein was studied using differentialscanning calorimetry and circular dichroism spectroscopy. Bothmethods yielded comparable values. The analysis revealed anunfolding enthalpy at 70°C of 70.2 ± 4.0 kcal/moland a     

Characterization of glycosylated variants of {beta}-lactoglobulin expressed in Pichia pastoris

Glycosylated variants of ß-lactoglobulin (BLG) wereproduced in the methylotrophic yeast Pichia pastoris to mimicthe glycosylation pattern of glycodelin, a homologue of BLGfound in humans. Glycodelin has three sites for glycosylation,corresponding to amino acids 63–65 (S1), 85–87 (S2)and 28–30 (S3) of BLG. These three sites were engineeredinto BLG to produce the variants S2, S12 and S123, which carriedone, two and three glycosylation sites, respectively. The oligosaccharideson these BLG variants ranged from (mannose)₉(N-acetylglucosamine)₂(Man₉GN₂) to Man₁₅GN₂ and were of the     

Engineering of multiple arginines into the Ser/Thr surface of Trichoderma reesei endo-1,4-{beta}-xylanase II increases the thermotolerance and shifts the pH optimum towards alkaline pH

We studied the effects of increase in the number of surfacearginines on the enzyme activity and stability of Trichodermareesei endo-1,4-ß-xylanase II. The number of arginineswas increased in two mutant series. The first set containedsix arginines on different sides of the protein surface. Thesearginines had no significant effect on the thermostability.However, the optimal pH region became narrower. Another seriesof five arginines was engineered into the `Ser/Thr surface',formed of part of the double-layered ß-sheet locatedon one side of the `right-hand-like' xylanase. These mutationsshifted the activity profile to the alkaline region by ~0.5–1.0pH units. In addition, the arginines on the Ser/Thr surfaceincreased the enzyme activity at high temperature, althoughthe enzyme stability in the absence of substrate decreased significantlyat 50–55°C. In the presence of the substrate, thethermostability increased 4–5-fold at 60–65°C.Thus, the substrate neutralized the destabilizing effect ofSer/Thr surface arginines and revealed a stabilizing effectof the same mutations. The stabilizing effect of arginines athigh temperatures was seen clearly only when five arginineswere introduced into the Ser/Thr surface.     

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.     

A new protein conjugate that replaces the use of secondary antibodies engineered from the two staphylococcal enzymes protein A and 6-phospho-{beta}-galactosidase

The lacG gene encoding the 6-phospho-ß-galactosidase(E.C.3.2.1.85) of Staphylococcus aureus was fused to the proteinA gene in the plasmid pRIT2T. Escherichia coli cells containingthis plasmid produce a fusion protein with both IgG bindingand 6-phospho-ß-galactosidase activities after heatinduction. The recombinant gene was overexpressed and the hybridprotein was purified to homogeneity in high yield. The chimericprotein was shown to have almost identical enzymatic characteristicsto pure 6-phospho-ß-galactosidase. This result leadsto the conclusion that a free N-terminus of the 6-phospho-ß-galactosidaseis not required for biological activity. The hybrid proteinof protein A and 6-phospho-ß-galactosidase was usedas an enzyme conjugate in enzyme-linked immunosorbent assays(ELISA). The experiments presented demonstrate that the 6-phospho-ß-galactosidaseis a suitable fusion partner in various diagnostic applicationswhere an unique biological activity is required.     

The role of residues outside the active site: structural basis for function of C191 mutants of Escherichia coli aspartate aminotransferase

In previous kinetic studies of Escherichia coli aspartate aminotransferase,it was determined that some substitutions of conserved cysteine191, which is located outside of the active site, altered thekinetic parameters of the enzyme (Gloss,L.M., Spencer,D.E. andKirsch,J.F., 1996, Protein Struct. Funct. Genet., 24, 195–208).The mutations resulted in an alkaline shift of 0.6–0.8pH units for the pK_a of the internal aldimine between the PLPcofactor and Lys258. The change in the pK_a affected the pH dependenceof the k_cat/K_m (aspartate) values for the mutant enzymes. Tohelp to understand these observations, crystal structures offive mutant forms of E.coli aspartate aminotransferase (themaleate complexes of C191S, C191F, C191Y and C191W, and C191Swithout maleate) were determined at about 2 Å resolutionin the presence of the pyridoxal phosphate cofactor. The overallthree-dimensional fold of each mutant enzyme is the same asthat of the wild-type protein, but there is a rotation of themutated side chain around its C