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.     

3-D structure of the D153G mutant of Escherichia coli alkaline phosphatase: an enzyme with weaker magnesium binding and increased catalytic activity

The substitution of aspartate at position 153 in Escherichiacoli alkaline phosphatase by glycine results in a mutant enzymewith 5-fold higher catalytic activity (k_cat but no change inKm at pH 8.0 in 50 mM Tris-HCl. The increased k_cat is achievedby a faster release of the phosphate product as a result ofthe lower phosphate affinity. The mutation also affects Mg²⁺binding, resulting in an enzyme with lower metal affinity. The3-D X-ray structure of the D153G mutant has been refined at2.5 Å to a crystallographic Rfactor of 16.2%. An analysisof this structure has revealed that the decreased phosphateaffinity is caused by an apparent increase in flexibility ofthe guanidinium side chain of Argl66 involved in phosphate binding.The mutation of Aspl53 to Gly also affects the position of thewater ligands of Mg²⁺, and the loop Glnl52–Thrl55 is shiftedby 0.3 Å away from the active site. The weaker Mg²⁺ bindingof the mutant compared with the wild type is caused by an alteredcoordination sphere in the proximity of the Mg²⁺ ion, and alsoby the loss of an electrostatic interaction (Mg²⁺.COO^-Aspl53)in the mutant Its ligands W454 and W455 and hydroxyl of Thrl55,involved in the octahedral coordination of the Mg²⁺ ion, arefurther apart in the mutant compared with the wild-type     

Protein secretion controlled by a synthetic gene in Escherichia coli

The inability of Escherichia coli to secrete proteins in growthmedium is one of the major drawbacks in its use in genetic engineering.A synthetic gene, homologous to the one coding for the kil peptideof pColE1, was made and cloned under the control of the lacpromoter, in order to obtain the inducible secretion of homologousor heterologous proteins by E.coli. The efficiency of this syntheticgene to promote secretion was assayed by analysing the productionand secretion of two proteins, the R-TEM1 ß-lactamase,and the -amylase from Bacillus licheniformis. This latter proteinwas expressed in E.coli from its gene either on the same plasmidas the kil gene or on a different plasmid. The primary effectof the induction of the kil gene is the overproduction of thesecreted proteins. When expressed at a high level, the kil genepromotes the overproduction of all periplasmic proteins andthe total secretion in the culture medium of both the ß-lactamaseor the -amylase. This secretion is semi-selective for most periplasmkproteins are not secreted. The kil peptide induces the secretionof homologous or heterologous proteins in two steps, first actingon the cytoplasmic membrane, then permeabilizing the outer membrane.This system, which is now being assayed at the fermentor scale,is the first example of using a synthetic gene to engineer anew property into a bacterial strain.     

Insertion of a disulfide-containing neurotoxin into E.coil alkaline phosphatase: the hybrid retains both biological activities

We have inserted a disulfide-containing snake neurotoxin intothe N-terminal end of Escherichia coli alkaline phosphatase,between residues +6 and +7 of the mature enzyme. For this purpose,we have designed a cloning and expression vector which allowsinsertion of foreign DNA between the corresponding codons, andvisual selection of the desired recombinant clones upon recoveryof phosphatase activity. The hybrid protein is exported to thebacterial periplasm, the alkaline phosphatase signal peptideis correctly processed, and both domains are functionally conformed.The phosphatase domain displays catalytic activity, and theinserted toxin is able to bind to its biological target, thenicotinic acetylcholine receptor. The hybrid molecule is remarkablystable and resistant to proteolysis. Crude periplasmic extractcontaining the hybrid can be used as a tracer-containing reagentin competitive enzymo-immuno and enzymo-receptor assays. Wepropose to use the system described in this paper for fast preparationof properly folded disulfide-containing enzymatic probes.     

Expression of catalytically active hamster dihydroorotase domain in Escherichia coli: purification and characterization

Dihydroorotase is the central domain of trifunctional L-dihydroorotatesynthetase which also contains carbamyl phosphate synthetaseat the N-terminus and aspartate transcarbamylase at the C-terminus.The cDNA, corresponding to the active dihydroorotase domainas isolated after digestion of dihydroorotate synthetase withelastase, has been sub-cloned into the expression vector pCW12which was then used to transform Escherichia coli SØ1263pyrC^– lacking dihydroorotase activity. However, inductionof this recomhinant strain with IPTG produced large amountsof the dihydroorotase domain which were completely inactive.A number of cDNAs were expressed which were longer on the C-terminalside; all cDNAs expressed active dihydroorotase domain downto a minimal extension of 12 ammo adds (-Val- Pro-Pro-Gly-Tyr-GIy-Gm-Asp-Val-Arg-Lys-Trp)into the bridge region between the dihydroorotase and aspartatetranscarbamylase domains. Part of this dodecapeptide may forman amphipathk helix which in some way constrains the isolated,recombinant dihydroorotase domain to an active conformation.The recombinant hamster dihydroorotase purified from a cell-freeextract of E.coli in four steps has a turnover number of 297mol/min/(mol domain) for the conversion of L-dihydroorotateback to N-carbamyl-Laspartate with K₈ = 8.7 ± 1.5 µMfor L-dihydroorotate, a subunit molecular weight of 39 008 determinedfrom the sequence and 37 900 ± 400 when subjected toSDS–PAGE, and an isoelectric point of 5.7. Ultracentrifugalanalysis of the recombinant domain showed a single species ofs_20,w = 4.1 S and a single molecular species of M_r = 76 000corresponding to a dimer.     

Greek key jellyroll protein motif design: expression and characterization of a first-generation molecule

A protein designed de novo to fold into the Greek key jellyrollstructural motif has been studied. Theoretical analyses haveindicated that the designed sequence should adopt the ß-strandarrangement of the Greek key jellyroll rather than any otherarrangement. A synthetic gene was constructed and the proteinexpressed in Escherichia coli. Circular dichroism spectroscopyis consistent with the protein folding into the designed conformationand also suggests the presence of tertiary structure. Fluorescencespectroscopy showed the single tryptophan to be partially buried,while denaturation studies showed changes in fluorescence toprecede alterations in secondary structure.     

Display of {beta}-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp'-OmpA'-{beta}-lactamase fusions

Bacterial cell-surface exposure of foreign peptides and solubleproteins has been achieved recently by employing a fusion proteinmethodology. An Lpp'–OmpA(46–159)–Bla fusionprotein has been shown previously to display the normally periplasmicenzyme ß-lactamase (Bla) on the cell surface of theGram-negative bacterium Escherichia coli. Here, we have investigatedthe role of the OmpA domain of the tripartite fusion proteinin the surface display of the passenger domain (Bla) and havecharacterized the effects of the fusion proteins on the integrityand permeability of the outer membrane. We show that in additionto OmpA(46–159), a second OmpA segment, consisting ofamino acids 46–66, can also mediate the display of Blaon the cell surface. Other OmpA domains of various lengths (aminoacids 46–84, 46–109, 46–128, 46–141and 46–145) either anchored the Bla domain on the periplasmicface of the outer membrane or caused a major disruption of theouter membrane, allowing the penetration of antibodies intothe cell. Detergent and antibiotic sensitivity and periplasmicleakage assays showed that changes in the permeability of theouter membrane are an unavoidable consequence of displayinga large periplasmic protein on the surface of E.coli. This isthe first systematic report on the effects that cell surfaceengineering may have on the integrity and permeability propertiesof bacterial outer membranes.     

Involvement of various amino- and carboxyl-terminal residues in the active site of the histidine-containing protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus carnosus: site-directed mutagenesis with the ptsH gene, biochemical characterization and NMR studies of the mutant proteins

The phosphocarrier HPr (heat stable protein) of Staphylococcuscarnosus was modified by site-directed mutagenesis of the correspondingptsH gene in order to analyse the importance of amino acidswhich were supposed to be part of the active centre of the protein.Three residues which are conserved in all HPrs, Argl7, Prol8and Glu84, were mutated: Argl7 was changed to His (17RH) andPro18 and Glu84 were changed into Ala (18PA and 84EA). In addition,Leu86 was changed into Ala (86LA) and one mutant protein wasmissing the last six residues of the HPr (83). The wild typegene and all mutant genes were overexpressed and the gene productspurified to homogeneity. Three-dimensional structures of wildtype and mutant proteins were monitored by NMR spectroscopy.All five mutant HPrs had native conformations. The ATP-dependentHPr kinase can phosphorylate all HPr derivatives at Ser46. ThePTS activity of the amino-terminal HPr mutant proteins 17RHand 18PA was different compared to wild type HPr. In contrast,the car boxy-terminal mutant HPrs possessed a similar enzymeactivity to the wild type HPr. The 17RH and 18PA HPrs with substitutionnear the active centre His15 showed a very slow phosphorylationby enzyme I but the further transfer of the phosphoryl groupto enzyme III was also strongly inhibited. The enzyme activityof the HPr 17RH was significantly improved at low pH. NMR pH-titrationexperiments showed that Arg17 is not responsible for the lowpK_a, of the active centre His15 but this positively chargedresidue is essential in this position for the HPr activity.     

Directed evolution of a type I antifreeze protein expressed in Escherichia coli with sodium chloride as selective pressure and its effect on antifreeze tolerance

Both freezing tolerance and NaCI tolerance are improved whenantifreeze proteins are expressed as fusion proteins with twodomains of staphylococcal protein A (SPA) in Escherichia coli.To characterize these properties further we created a randomlymutated expression library in E.coli, based on the winter flounderantifreeze protein HPLC-8 component gene. Low-fidelity PCR productsof this gene were fused to the spa gene encoding two domainsof the SPA. The library was screened for enhanced NaCl toleranceand four clones were selected. The freezing tolerance of eachof the selected clones was enhanced to varying extents. DNAsequencing of the isolated mutants revealed that the amphiphilicproperties of the native antifreeze protein were essentiallyconserved. Furthermore, by studying the primary sequence ofthe randomly mutated clones, in comparison with the degree offreezing tolerance, we have identified clues which help in understandingthe relationship between salt and freezing tolerance.     

Synthesis, purification and initial structural characterization of octarellin, a de novo polypeptide modelled on the {alpha}/{beta}-barrel Proteins

We have attempted to construct an artificial polypeptide thatfolds like the eight-stranded parallel ß-barrel structures.Our approach consists of repeating eight times a unit peptidedesigned to adopt a ‘ß-strand/-helix’pattern. A first ‘test’ sequence for this structuralunit was deduced from a series of parameters defined after ananalysis of three natural /ß-barrel proteins and includingprincipally the lengths of the secondary structure elements,the /ß packing and the fitting on average Garnierprofiles. The gene encoding this structural unit was synthesized,cloned and expressed in Escherichia coli either as a monomeror as direct repeats of 2–12 units. Preliminary structuralcharacterization of the 7-, 8- and 9-fold unit polypeptidesby circular dichroism measurements indicates the presence ofthe predicted amount of -helix in the three proteins. Furtheranalysis by urea-gradient gel electrophoresis demonstrates that,in the conditions tested, only the 8-fold unit polypeptide formsa compact structure through a cooperative and rapid two-statefolding transition involving long-range molecular interactions.     

Crystallization and structure solution at 4 A resolution of the recombinant synthase domain of N(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli complexed to a substrate analogue

The recombinant synthase domain of the bifunctional enzyme N-(5'-phosphoribosyl)anthranilateisomerase:indole-3-glycerol-phosphate synthase from Escherichiacoli has been crystallized, and the structure has been solvedat 4 Å resolution. Two closely related crystal forms grownfrom ammonium sulphate diffract to 2 Å resolution. Oneform (space group R32, a = 163 Å, = 29.5°) containsthe unliganded synthase domain; the second crystal form (spacegroup P6₃22, a = 144 Å, c = 158 Å) is co-crystallizedwith the substrate analogue N-(5'-phosphoribit-1-yl)anthranilate.The structure of the synthase–inhibitor complex has beensolved by the molecular replacement method. This achievementrepresents the first successful use of a (ß)_g-barrelmonomer as a trial model. The recombinant synthase domain associatesas a trimer in the crystal, the molecules being related by apseudo-crystallographic triad. The interface contacts betweenthe three domains are mediated by those residues that are alsoinvolved in the domain interface of the bifunctional enzyme.This system provides a model for an interface which is usedin both intermolecular and intramolecular domain contacts.     

Chemical synthesis in protein engineering: total synthesis, purification and covalent structural characterization of a mitogenic protein, human transforming growth factor-alpha

Successful approaches to protein engineering required that thedesired analogs be easily and rapidly obtained in sufficientquantities and purities for unambiguous structural and functionalcharacterizations. Chemical synthesis is the method of choicefor engineering small peptides. We now demonstrate that withimproved methodologies and instrumentation, total chemical synthesiscan be used to produce a small protein in a form suitable forengineering studies. Active human transforming growth factor-alpha(TGF-), a 50 amino acid long protein with three disulfide bonds,has been synthesized and purified in multiple tens of mg amountsin <7 days. The purified human TGF- migrated as a singleband on SDS–polyacrylamide gels, ran as a single sharpmajor band at pI = 6.2 on isoelectric focusing gels, displayedan MW = 5546.2 (Th.5546.3) by mass spectrometry, contained threedisulfide bonds and had EGF receptor binding, mitogenic andsoft agar colony formation activities. The locations of disulfidebonds were found to be analogous to those found in epidermalgrowth factor (EGF) and in human TGF- expressed in bacteria.