Portal de Periódicos da CAPES

Replacement of a Phenylalanine by a Tyrosine in the Active Site Confers Fructose-6-phosphate Aldolase Activity to the Transaldolase of Escherichia coli and Human Origin

2008; Elsevier BV; Volume: 283; Issue: 44 Linguagem: Inglês

10.1074/jbc.m803184200

1083-351X

Sarah Schneider, Tatyana Sandalova, G. Schneider, Georg A. Sprenger, Anne K. Samland,

Porphyrin Metabolism and Disorders

Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe178 to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody. Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe178 to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody. Transaldolase (Tal) 2The abbreviations used are: Tal, transaldolase; DHA, dihydroxyacetone; Ery-4-P, d-erythrose 4-phosphate; Fru-6-P, d-fructose 6-phosphate; FSA, fructose-6-phosphate aldolase; GAP, dl-glyceraldehyde 3-phosphate; GST, glutathione S-transferase; hTal, human transaldolase (Taldol). is a ubiquitous enzyme that is present in all domains of life. It is part of the non-oxidative path of the pentose phosphate pathway. Here, it catalyzes the reversible transfer of a dihydroxyacetone (DHA) moiety from a ketose donor, e.g. fructose 6-phosphate (Fru-6-P), onto an aldehyde acceptor, e.g. erythrose 4-phosphate (Ery-4-P). The best studied example is transaldolase B (TalB) from Escherichia coli (1Sprenger G.A. Schörken U. Sprenger G. Sahm H. J. Bacteriol. 1995; 177: 5930-5936Crossref PubMed Google Scholar). A number of structural and mechanistic studies have been published elucidating its reaction mechanism (1Sprenger G.A. Schörken U. Sprenger G. Sahm H. J. Bacteriol. 1995; 177: 5930-5936Crossref PubMed Google Scholar, 2Schneider G. Sprenger G.A. Methods Enzymol. 2002; 354: 197-201Crossref PubMed Scopus (7) Google Scholar, 3Schörken U. Jia J. Sahm H. Sprenger G.A. Schneider G. FEBS Lett. 1998; 441: 247-250Crossref PubMed Scopus (14) Google Scholar, 4Schörken U. Thorell S. Schürmann M. Jia J. Sprenger G.A. Schneider G. Eur. J. Biochem. 2001; 268: 2408-2415Crossref PubMed Scopus (35) Google Scholar, 5Jia J. Huang W. Schörken U. Sahm H. Sprenger G.A. Lindqvist Y. Schneider G. Structure. 1996; 4: 715-724Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 6Jia J. Schörken U. Lindqvist Y. Sprenger G.A. Schneider G. Protein Sci. 1997; 6: 119-124Crossref PubMed Scopus (64) Google Scholar). Similar to class I aldolases the reaction proceeds via a Schiff base intermediate. TalB is a homo-dimer and the monomer exhibits a (β/α)8-barrel fold where the C-terminal helix lies across the barrel opening at one site (5Jia J. Huang W. Schörken U. Sahm H. Sprenger G.A. Lindqvist Y. Schneider G. Structure. 1996; 4: 715-724Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). A similar structure had been determined for the human transaldolase (7Thorell S. Gergely Jr., P. Banki K. Perl A. Schneider G. FEBS Lett. 2000; 475: 205-208Crossref PubMed Scopus (35) Google Scholar). Recently, a fructose-6-phosphate aldolase (FSA) of E. coli has been discovered by our group (8Schürmann M. Sprenger G.A. J. Biol. Chem. 2001; 276: 11055-11061Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) that uses DHA as donor substrate and catalyzes the reversible formation of Fru-6-P from DHA and GAP (Fig. 1). Multiple sequence alignments of different Tal sequences demonstrate that FSA resides within the family of transaldolases (8Schürmann M. Sprenger G.A. J. Biol. Chem. 2001; 276: 11055-11061Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 9Thorell S. Schürmann M. Sprenger G.A. Schneider G. J. Mol. Biol. 2002; 319: 161-171Crossref PubMed Scopus (66) Google Scholar) and does show little similarity to DHAP-dependent aldolases, such as class I fructose-1,6-bisphosphate aldolase. Also the FSA monomer is highly similar to TalB exhibiting a similar (β/α)8-barrel fold (9Thorell S. Schürmann M. Sprenger G.A. Schneider G. J. Mol. Biol. 2002; 319: 161-171Crossref PubMed Scopus (66) Google Scholar). But in contrast to TalB, FSA forms a homo-decamer, which is arranged by two doughnut-shaped pentameric rings where the C-terminal helix of one subunit interacts with the adjacent subunit (9Thorell S. Schürmann M. Sprenger G.A. Schneider G. J. Mol. Biol. 2002; 319: 161-171Crossref PubMed Scopus (66) Google Scholar). With 220 amino acids compared with 317 amino acids FSA is smaller than TalB and shows about 20% sequence identity to TalB. Mechanistically, it is a class I aldolase, i.e. the reaction proceeds via a Schiff base intermediate. In this study, we addressed the differences between Tal and FSA concerning their enzymatic features especially the Fru-6-P cleavage. Therefore, we compared TalB and FSA from E. coli using a structure-assisted sequence alignment (Fig. 2) to identify differences in sequence in or close to the active site of TalB. We chose 11 positions at which site-saturation mutagenesis libraries were created. Screening of these libraries for Fru-6-P formation from DHA and GAP was performed using a newly developed color assay. One replacement, Phe178 to Tyr, resulted in a strongly elevated FSA activity and we here report a detailed functional and structural characterization of the human and E. coli Tal F189Y and F178Y variants, respectively. The described muteins are examples of a change in enzyme class from a transferase to a lyase conferred by a single amino acid replacement. Chemicals and Auxiliary Enzymes—Antibiotics, acrylamide-bisacrylamide, sugars, buffer components, and culture media for bacteria were from Carl Roth GmbH (Karlsruhe, Germany). Restriction enzymes were purchased from New England Biolabs (Frankfurt, Germany) and MBI fermentas (St. Leon-Rot, Germany). Sugar phosphates and aldehydes were from Sigma/Fluka (Taufkirchen, Germany). Auxiliary enzymes were from Roche Applied Science (Mannheim, Germany). Q-Sepharose and gel filtration matrix Superdex 200pg were from Amersham Biosciences/GE Healthcare, histidine-tagged affinity column matrix nickel-nitrilotriacetic acid was purchased from Qiagen (Hilden, Germany). Strains and Plasmids—Bacterial strains, plasmids, and oligonucleotides used in this study are listed in supplementary data Tables S1 and S2. Site-directed Saturation Mutagenesis—The talB gene was amplified by PCR from chromosomal DNA of E. coli W3110 and cloned into the pET28a vector. During this amplification 4 bases at positions 726 (G–T), 738 (G–T), 739 (G–A), and 759 (G–T) were changed as compared with the sequence in the data base (10Hayashi K. Morooka N. Yamamoto Y. Fujita K. Isono K. Choi S. Ohtsubo E. Baba T. Wanner B.L. Mori H. Horiuchi T. Mol. Syst. Biol. 2006; 2 (2006): 0007Crossref PubMed Scopus (364) Google Scholar). This results inadvertently in an amino acid replacement of Ala by Thr at position 247. The NdeI-XhoI fragment of pET28talB was cloned into expression vector pJF119EH (11Fürste J.P. Pansegrau W. Frank R. Blöcker H. Scholz P. Bagdasarian M. Lanka E. Gene (Amst.). 1986; 48: 119-131Crossref PubMed Scopus (813) Google Scholar). This fragment contained the ribosomal binding site of the pET28a vector, an N-terminal His6 tag, and the talB gene of E. coli W3110. Site-directed saturation mutagenesis was performed using the QuikChange® II Site-directed Mutagenesis Kit from Stratagene (Amsterdam, The Netherlands) and degenerated oligonucleotides (biomers, Ulm, Germany) with NNS degeneracy (N, A/C/G/T; S, C/G). Thereby, 32 codons and all 20 proteinogenic amino acid residues were obtained. The libraries were transformed into XL1Blue cells (Stratagene). The plasmid DNA of 30 randomly picked clones of one library (Phe178X) were isolated with the NucleoSpin Kit II (Macherey + Nagel, Düren, Germany) and custom sequenced by MWG Biotech AG (Martinsried, Germany). For 10 randomly picked clones the expression in crude extracts was checked via SDS-PAGE. All of them expressed the corresponding His6-TalBF178X variant protein in considerable amounts. Cultivation and Protein Expression in Deepwell Plates—Single colonies were randomly picked and arrayed in 96-deepwell plates (volume 2 ml; Greiner Bio-One, Frickenhausen, Germany). Each well contained 1 ml of 2YT broth (12Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) with 100 μg/ml ampicillin. The plate was sealed with a gas-permeable adhesive film and incubated at 37 °C with shaking at 145 rpm overnight. These cultures were used to inoculate expression cultures (1:20) containing 1 ml of 2YT broth supplemented with 100 μg/ml ampicillin and 1 mm (final concentration) isopropyl β-d-thiogalactopyranoside. These expression cultures were grown at 37 °C with shaking at 145 rpm overnight. The cells were collected by centrifugation (1600 × g at 8 °C for 10 min), supernatants were discarded. Cell pellets were resuspended in 100 μl of 50 mm glycylglycine buffer (pH 8.5) containing 1 mg/ml lysozyme (Roche) and 0.1 mg/ml DNase I (Roth). Cell debris was collected by centrifugation (1600 × g at 8 °C for 10 min) and 100 μl of the cell-free extracts were transferred to a new 96-well microtiter plate for enzyme assay. Color Assay for FSA Activity—The previously described assay for fructose 6-phosphate formation (8Schürmann M. Sprenger G.A. J. Biol. Chem. 2001; 276: 11055-11061Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) was adapted for screening in a microtiter plate format. The formation of Fru-6-P was coupled to the reduction of NADP+ via phosphoglucose isomerase and glucose-6-phosphate dehydrogenase. NADPH was visualized by the formation of a lilac formazan complex. This allowed a visual detection of the appearance of positive hits. For quantitative analysis the increase in color was monitored for 20 min at 540 nm at 25 °C using a multiwell photometer (Spectra Max Plus, Molecular Devices, Ismaning/Munich, Germany). The assay contained 50 mm glycylglycine buffer (pH 8.5), DHA (50 mm), dl-glyceraldehyde 3-phosphate (GAP, 2.8 mm), NADP+ (0.5 mm), phosphoglucose isomerase (0.1 unit), glucose-6-phosphate dehydrogenase (0.1 unit), nitro tetrazolium blue chloride (0.5 mm), and diaphorase (0.3 milliunit, Sigma). The reaction was started by adding cell-free extract (≤5 μg of protein). Protein concentrations were measured in microtiter plates using a dye-binding method (13Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216377) Google Scholar). The activities were monitored as an increase in OD540 per min and standardized over the protein concentration using Microsoft Excel XP (Unterschleißheim, Germany). Cell-free extracts containing FSA and His6-TalBwt were used as positive and negative controls, respectively. The standard deviations of the activities of the positive and negative controls were <20%. Protein Purification of His6-TalBwt and His6-TalBF178Y—From cell-free extracts of the E. coli XL1Blue recombinant strains (XL1Blue-pJF119talB and XL1Blue-pJF119talBF178Y), His6-TalB and His6-TalBF178Y were purified by nickel-nitrilotriacetic acid affinity chromatography as recommended by the supplier (Qiagen, The QIAexpressionist™, 5th edition). Fractions containing transaldolase activity were pooled and the buffer was changed to 50 mm glycylglycine (pH 8.5) containing 1 mm dithiothreitol using a concentrator unit (Vivaspin MWC 10,000 Da; Vivascience, Göttingen, Germany). The purity was estimated by SDS-PAGE (14Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar). Purification of FSA—From cell-free extracts of recombinant strain BL21(DE3) Star pLysS-pET16fsa, FSA was purified as described previously (15Inoue T. Microbial Aldolases as C-C Bonding Enzymes: Investigations of Structural-Functional Characteristics and Application for Stereoselective Reactions. Ph.D. thesis, Universität Stuttgart, Stuttgart, Germany2006Google Scholar). After heat treatment (20 min, 75 °C) and centrifugation (100,000 × g at 4 °C for 35 min) the supernatant was applied to an anion exchange column (Q-Sepharose HP (16/10)). The FSA activity containing fractions were pooled and buffer was exchanged to 50 mm glycylglycine buffer (pH 8.0) containing 1 mm dithiothreitol. The protein purity was determined by SDS-PAGE and estimated to ≥98% purity. Activity Assays—All enzyme assays were performed in 50 mm glycylgycine buffer (pH 8.5) containing 1 mm dithiothreitol at 30 °C. Using a Cary 100 Bio UV-Visible Spectrophotometer (Varian, Darmstadt, Germany) the enzyme activity was monitored at 340 nm for 10 min. For the transaldolase reaction d-fructose 6-phosphate (10 mm) and d-erythrose 4-phosphate (2 mm according to 61% purity of the supplied material) were used as substrates. The formation of GAP was detected via the formation of NAD+ from NADH using the coupling enzymes triose-phosphate isomerase and glycerol-3-phosphate dehydrogenase. The assay was performed as described (1Sprenger G.A. Schörken U. Sprenger G. Sahm H. J. Bacteriol. 1995; 177: 5930-5936Crossref PubMed Google Scholar, 16Tsolas O. Horecker B.L. The Enzymes, 3rd Ed. Academic Press, New York1972Google Scholar). To distinguish between the Fru-6-P cleavage activity and Tal activity the reaction was started with Ery-4-P. The decrease of NADH in the presence of Fru-6-P was subtracted from the decrease in the presence of both substrates, Fru-6-P and Ery-4-P. For determination of the Km value for d-fructose 6-phosphate a concentration range of 1 to 50 mm (His6-TalBwt), 1 to 150 mm (His6-TalBF178Y), and 0.5 to 60 mm (hTalwt, hTalF89Y) were used at a constant concentration of d-erythrose 4-phosphate (2 mm). This concentration of d-erythrose 4-phosphate is saturating as the Km value for TalB is 90 μm (1Sprenger G.A. Schörken U. Sprenger G. Sahm H. J. Bacteriol. 1995; 177: 5930-5936Crossref PubMed Google Scholar). The specific activity was plotted against the Fru-6-P concentration. The hyperbolic function of the Michaelis-Menten equation was fitted to the data using SigmaPlot 9.0 (Systat Software, Erkrath, Germany). The cleavage of d-fructose 6-phosphate into dihydroxyacetone and d-glyceraldehyde 3-phosphate was monitored via an enzyme coupled assay as described previously (8Schürmann M. Sprenger G.A. J. Biol. Chem. 2001; 276: 11055-11061Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Again, the formation of GAP is detected. The Km value for d-fructose 6-phosphate was determined using concentrations of d-fructose 6-phosphate ranging from 1 to 50 mm (FSAwt), 1 to 20 mm (His6-TalBF178Y), or 0.25 to 60 mm (hTalF189Y). The formation of d-fructose 6-phosphate from dihydroxyacetone and dl-glyceraldehyde 3-phosphate was determined as described previously (8Schürmann M. Sprenger G.A. J. Biol. Chem. 2001; 276: 11055-11061Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) via the coupling enzymes phosphoglucose isomerase and glucose-6-phosphate dehydrogenase. The Km value for dihydroxyacetone was determined using 10 to 300 mm (FSAwt), 10 to 350 mm (His6-TalBF178Y), or 10 to 600 mm (hTalF189Y). The concentration of dl-glyceraldehyde 3-phosphate was kept constant at 2.8 mm. This concentration of dl-glyceraldehyde 3-phosphate was saturating as the Km value for TalB was 38 μm (1Sprenger G.A. Schörken U. Sprenger G. Sahm H. J. Bacteriol. 1995; 177: 5930-5936Crossref PubMed Google Scholar). Gel Filtration—The molecular mass of His6-TalBF178Y and hTalF189Y under native conditions was determined by size exclusion chromatography. Gel filtration was performed on a Superdex 200pg (10/30) column and a fast protein liquid chromatography system (Amersham Biosciences). The column was equilibrated with 50 mm glycylglycine buffer (pH 8.5) containing 150 mm sodium chloride and 1 mm dithiothreitol with a flow rate of 0.5 ml/min at 4 °C. The column was calibrated with reference proteins of known molecular masses between 13.7 and 440 kDa (ribonuclease A, 13.7 kDa; chymotrypsinogen A, 25 kDa; ovalbumin, 43 kDa; albumin, 67 kDa; aldolase, 158 kDa; catalase, 232 kDa; ferritin, 440 kDa; purchased from GE Healthcare). The void volume was determined with blue dextran 1000. Crystallization and Structure Determination—Crystals of the E. coli TalB mutant F178Y were obtained at room temperature by the hanging-drop vapor diffusion method at several conditions, all conditions contained polyethylene glycol and different salts. The best diffracting crystals were obtained in 18% polyethylene glycol 3350, 0.2 m ammonium sulfate, without addition of any buffer. Two μl of a 60 mg/ml protein solution in 50 mm glycylglycine (pH 8) were mixed with 4 μl of the crystallization reservoir solution and equilibrated against 1 ml of mother liquor. Crystals grew during several days to a size of 0.2 × 0.1 × 0.05 mm. X-ray data were collected at beamline ID14-3 (ESRF Grenoble, France) (λ = 0.931 Å) using an ADSC Q4 CCD detector. The crystals were soaked for several seconds in crystallization solution containing 25% glycerol before freezing in a stream of liquid nitrogen. Crystals of the F178Y mutant belong to the same orthorhombic space group P212121 as wild-type protein with slightly different cell dimensions a = 65.9 Å, b = 86.2 Å, c = 131.2 Å. All diffraction data were processed with the program MOSFLM, scaled, and merged with SCALA from the CCP4 suite of programs (17Collaborative Computational Project N. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19768) Google Scholar). The data collection statistics are given in Table 1.TABLE 1Data collection and refinement statisticsData collectionSpace groupP212121Unit cell (Å)a = 65.9, b = 86.2, c = 131.2Resolution (Å)47-1.4 (1.48-1.4)No. of unique reflections135,268 (13,203)Rmerge0.037 (0.15)Redundancy3.7 (2.4)Mean I/σ(I)21.4 (5.7)Completeness92.0% (62%)RefinementR/Rfree14.9/17.3No. of protein atoms5,116No. of ions4No. of water molecules605Root mean square deviation bonds (Å)0.09Root mean square deviation angles (°)1.2Average B-factor (Å)2Protein11.8Ions15.5Water21.7Ramachandran plotMost favoured (%)98.2Allowed1.6Outliers0.2 Open table in a new tab The structure of the F178Y mutant was solved by molecular replacement with MOLREP (18Vagin A.A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4152) Google Scholar) using a subunit of wild-type E. coli TalB (5Jia J. Huang W. Schörken U. Sahm H. Sprenger G.A. Lindqvist Y. Schneider G. Structure. 1996; 4: 715-724Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) (Protein Data Bank 1ONR) as a template. Estimation of the solvent content in the crystals of F178Y suggested that the asymmetric unit contains two subunits. The native Patterson map shows a strong peak (50% of the origin peak) at 0.0, 0.5, and 0.076 indicating the presence of translational symmetry. The best solution found with MOLREP had an R-factor of 0.434 and a score of 0.75. Refinement of the model using REFMAC5 (19Murshudov G. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-253Crossref PubMed Scopus (13869) Google Scholar) was monitored by Rfree and alternated with manual inspection and rebuilding in COOT (20Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23375) Google Scholar). The final model contains 631 amino acid residues (residues 2–317 for chain A and 3–317 for chain B), 4 sulfate ions, and 717 water molecules. Refinement statistics are shown in Table 1. The crystallographic data for the F178Y mutant has been deposited with the Protein Data Bank accession code 3CWN. Cloning and Mutagenesis of the Human taldo1 Gene—The full-length cDNA clone IRATp970D0610D was obtained from the RZPD data base (Deutsches Ressourcenzentrum für Genomforschung GmbH, Berlin, Germany). The coding sequence for the human transaldolase Taldo1 (hTal) (21Banki K. Halladay D. Perl A. J. Biol. Chem. 1994; 269: 2847-2851Abstract Full Text PDF PubMed Google Scholar) was amplified by PCR and cloned into expression vector pGEXTN* using restriction sites NdeI and SmaI. This allows the co-transcription and translation as GST fusion protein. The coding sequence was verified by sequencing. However, a comparison with the taldo1 sequence (21Banki K. Halladay D. Perl A. J. Biol. Chem. 1994; 269: 2847-2851Abstract Full Text PDF PubMed Google Scholar) revealed a deletion of 4 bases at position 829 bp resulting in a frameshift. Site-directed mutagenesis for correction of this deletion and replacement of Phe189 by Tyr was carried out using a modified version of the QuikChange® protocol. In this case the ReproFast Polymerase from GENAXXON (Biberach, Germany) was used. The correct sequence was confirmed by custom sequencing (MWG Biotech AG). Heterologous Expression and Purification of Human Tal—GST-hTal and GST-hTalF189Y were expressed in an E. coli LJ110 (DE3) talA-talB- pLysSRARE recombinant strain (LJ110-pGEXTN*htal and LJ110-pGEXTN*htalF189Y). This strain containing the DE3 system (22Rosenberg A.H. Lade B.N. Chui D.S. Lin S.W. Dunn J.J. Studier F.W. Gene (Amst.). 1987; 56: 125-135Crossref PubMed Scopus (1045) Google Scholar) was deficient in both genes encoding for transaldolase, talA and talB, 3A. K. Samland and G. A. Sprenger, unpublished results. and carried the pLysSRARE2 plasmid (23Novy R. Drott D. Yaeger K. Mierendorf R. inNovations. 2001; 12: 1-3Google Scholar). The chromosomal deletion of talA and talB was performed by the method described by Datsenko and Wanner (24Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11201) Google Scholar) in the E. coli strain BW25113. Using P1 phage transduction (12Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) it was transferred to E. coli LJ110 (DE3) strain. hTal and hTalF189Y were purified by a glutathione-Sepharose column as recommended by the supplier (GE Healthcare). The GST tag was cleaved off using TEV-Protease (25Parks T.D. Howard E.D. Wolpert T.J. Arp D.J. Dougherty W.G. Virology. 1995; 210: 194-201Crossref PubMed Scopus (98) Google Scholar) in an on-column digest overnight. The buffer was changed to 50 mm glycylglycine (pH 8.5) containing 1 mm dithiothreitol using a concentrator unit (Vivaspin MWC 10,000 Da; Vivascience, Göttingen, Germany). Immunodetection—5- and 15-μg cell-free extracts expressing TalB or GST-hTal variants, respectively, were separated by 12% (v/v) SDS-PAGE and stained with Coomassie Brilliant Blue. A second gel was run in parallel with 5 and 20 μg of cell-free extract and the proteins were transferred to a polyvinylidene fluoride membrane for immunodetection (Roche). Western blotting was done by standard procedures. The blotted transaldolase proteins were decorated with a polyclonal antibody against TalB from guinea pig (4Schörken U. Thorell S. Schürmann M. Jia J. Sprenger G.A. Schneider G. Eur. J. Biochem. 2001; 268: 2408-2415Crossref PubMed Scopus (35) Google Scholar). Antiguinea pig antibody coupled to alkaline phosphatase (Sigma) was used as second antibody and color development with nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate p-toluidine salt was monitored. Prestained all blue SDS-PAGE standard proteins (Bio-Rad) were used as marker proteins. Generation and Screening of Mutant Libraries—Based on a structure-assisted sequence alignment (Fig. 2) 11 positions were identified in or close to the active site of FSA and TalB from E. coli. At these positions both enzymes differ in their primary sequence. We reasoned that by changing these residues it was possible to confer an aldolase activity upon TalB. Therefore, at the following positions a site-saturation library was generated using talB as template: Ser94, Glu96, Ser135, Asn154, Leu157, Phe159, Ser176, Phe178, Gly180, Thr220, and Arg228. By applying a newly developed color assay (Fig. 3) it was possible to screen hundreds of clones in microtiter plates for the formation of Fru-6-P from DHA and GAP as substrate. DHA is a very poor substrate for TalBwt (Table 2).TABLE 2Kinetics for His6-TalBF178Y from E. coli, and hTalF189Y from H. sapiens sapiens as well as FSA from E. coli for the formation and cleavage of d-fructose 6-phosphateProteinHis6-TalBwtHis6-TalBF178YFSAhTalwthTalF189YReactionFru-6-P synthesis fromVmax (units/mg)NDaND, not determined; activity ≤0.1 unit/mg using 50 mm DHA and 2.8 mm dl-GAP7 ± 1bAverage value from four independent measurements20 ± 1bAverage value from four independent measurementsN.detect.cN. detect., not detectable; activity <0.01 units/mg14dAverage value from two independent measurements (deviation ≤14%)DHA and GAPkcatekcat was calculated as turnover number per active sites, i.e. monomeric subunits (s–1)ND4.3 ± 0.7bAverage value from four independent measurements7.6 ± 0.5N.detect.8.9dAverage value from two independent measurements (deviation ≤14%)Km (DHA) (mm)ND30 ± 4bAverage value from four independent measurements62 ± 7bAverage value from four independent measurementsN.detect.340dAverage value from two independent measurements (deviation ≤14%)kcat/Km (m–1 s–1)ND150 ± 30130 ± 8N.detect.26Fru-6-P cleavage intoVmax (units/mg)N.detect.0.36 ± 0.05bAverage value from four independent measurements3.4 ± 0.7bAverage value from four independent measurementsN.detect.0.32 ± 0.04fAverage value from three independent measurementsDHA and GAPkcatekcat was calculated as turnover number per active sites, i.e. monomeric subunits (s–1)N.detect.0.22 ± 0.03bAverage value from four independent measurements1.3 ± 0.3N.detect.0.21 ± 0.02fAverage value from three independent measurementsKm (F6P) (mm)N.detect.1.5 ± 0.2bAverage value from four independent measurements12 ± 3bAverage value from four independent measurementsN.detect.0.76 ± 0.11fAverage value from three independent measurementskcat/Km (m–1 s–1)N.detect.150 ± 26130 ± 35N.detect.270 ± 11a ND, not determined; activity ≤0.1 unit/mg using 50 mm DHA and 2.8 mm dl-GAPb Average value from four independent measurementsc N. detect., not detectable; activity <0.01 units/mgd Average value from two independent measurements (deviation ≤14%)e kcat was calculated as turnover number per active sites, i.e. monomeric subunitsf Average value from three independent measurements Open table in a new tab In a first round one plate of each focused library was screened, corresponding to 84 clones. Only the focused library at position Phe178 contained active clones. To ensure that all possible 20 amino acid replacements were covered a total of 252 clones were screened for the focused library Phe178X. Clones exhibiting an activity at least twice as high as the background (negative control, His6-TalBwt) were re-cultivated and re-screened in deepwell plates. Of 252 clones 19 exhibiting consistently FSA activity were sequenced. All of them possessed a TAC triplet instead of TTT coding for Tyr instead of Phe. No additional mutations had occurred. These results demonstrate that of the 11 positions chosen for the generation of focused libraries only position Phe178 is critical for gain of a fructose-6-phosphate aldolase activity. Furthermore, nature selected in FSA the best and only possible amino acid residue for conferring this activity. Purification and Quaternary Structure of the His6-TalBF178Y Mutein—To characterize the TalBF178Y variant in more detail the mutant protein (mutein) was purified to an estimated purity of 97% (Fig. 4). From 1 g wet E. coli cells about 10 and 9 mg of His6-TalBwt and His6-TalBF178Y mutein, respectively, were recovered. FSA and TalB exhibit a similar subunit structure of a (β/α)8-barrel fold but differ in their quarternary structure. FSA is a homo-decamer (9Thorell S. Schürmann M. Sprenger G.A. Schneider G. J. Mol. Biol. 2002; 319: 161-171Crossref PubMed Scopus (66) Google Scholar), whereas TalB is a homo-dimer (5Jia J. Huang W. Schörken U. Sahm H. Sprenger G.A. Lindqvist Y.