Title: Conversion of Aspartate Aminotransferase into anl-Aspartate β-Decarboxylase by a Triple Active-site Mutation
Abstract: The conjoint substitution of three active-site residues in aspartate aminotransferase (AspAT) of Escherichia coli (Y225R/R292K/R386A) increases the ratio ofl-aspartate β-decarboxylase activity to transaminase activity >25 million-fold. This result was achieved by combining an arginine shift mutation (Y225R/R386A) with a conservative substitution of a substrate-binding residue (R292K). In the wild-type enzyme, Arg386 interacts with the α-carboxylate group of the substrate and is one of the four residues that are invariant in all aminotransferases; Tyr225 is in its vicinity, forming a hydrogen bond with O-3′ of the cofactor; and Arg292interacts with the distal carboxylate group of the substrate. In the triple-mutant enzyme, k cat′ for β-decarboxylation of l-aspartate was 0.08 s−1, whereas k cat′ for transamination was decreased to 0.01 s−1. AspAT was thus converted into an l-aspartate β-decarboxylase that catalyzes transamination as a side reaction. The major pathway of β-decarboxylation directly produces l-alanine without intermediary formation of pyruvate. The various single- or double-mutant AspATs corresponding to the triple-mutant enzyme showed, with the exception of AspAT Y225R/R386A, no measurable or only very low β-decarboxylase activity. The arginine shift mutation Y225R/R386A elicits β-decarboxylase activity, whereas the R292K substitution suppresses transaminase activity. The reaction specificity of the triple-mutant enzyme is thus achieved in the same way as that of wild-type pyridoxal 5′-phosphate-dependent enzymes in general and possibly of many other enzymes, i.e. by accelerating the specific reaction and suppressing potential side reactions. The conjoint substitution of three active-site residues in aspartate aminotransferase (AspAT) of Escherichia coli (Y225R/R292K/R386A) increases the ratio ofl-aspartate β-decarboxylase activity to transaminase activity >25 million-fold. This result was achieved by combining an arginine shift mutation (Y225R/R386A) with a conservative substitution of a substrate-binding residue (R292K). In the wild-type enzyme, Arg386 interacts with the α-carboxylate group of the substrate and is one of the four residues that are invariant in all aminotransferases; Tyr225 is in its vicinity, forming a hydrogen bond with O-3′ of the cofactor; and Arg292interacts with the distal carboxylate group of the substrate. In the triple-mutant enzyme, k cat′ for β-decarboxylation of l-aspartate was 0.08 s−1, whereas k cat′ for transamination was decreased to 0.01 s−1. AspAT was thus converted into an l-aspartate β-decarboxylase that catalyzes transamination as a side reaction. The major pathway of β-decarboxylation directly produces l-alanine without intermediary formation of pyruvate. The various single- or double-mutant AspATs corresponding to the triple-mutant enzyme showed, with the exception of AspAT Y225R/R386A, no measurable or only very low β-decarboxylase activity. The arginine shift mutation Y225R/R386A elicits β-decarboxylase activity, whereas the R292K substitution suppresses transaminase activity. The reaction specificity of the triple-mutant enzyme is thus achieved in the same way as that of wild-type pyridoxal 5′-phosphate-dependent enzymes in general and possibly of many other enzymes, i.e. by accelerating the specific reaction and suppressing potential side reactions. In the engineering of protein catalysts with new functional properties, the modification of existing enzymes provides an alternative to the production of catalytic antibodies or, in a more distant future, the de novo design of enzymes. Enzyme engineering may be expected to contribute to elucidating both the structural basis of the functional properties and the course of the molecular evolution. Several attempts to change the substrate specificity of an enzyme by substitution of the substrate-binding residues have succeeded (Refs. 1Wilks H.M. Hart K.W. Freeney R. Dunn C.R. Muirhead H. Chia W.N. Barstow D.A. Atkinson T. Clarke A.R. Holbrook J.J. Science. 1988; 242: 1541-1544Crossref PubMed Scopus (246) Google Scholar, 2Bradley M. Bücheler U.S. Walsh C.T. Biochemistry. 1991; 30: 6124-6127Crossref PubMed Scopus (38) Google Scholar, 3Scutton N.S. Berry A. Perham R.N. Nature. 1990; 343: 38-43Crossref PubMed Scopus (646) Google Scholar, 4Hedstrom L. Szilagyi L. Rutter W.J. Science. 1992; 255: 1249-1253Crossref PubMed Scopus (452) Google Scholar, 5Onuffer J.J. Kirsch J.F. Protein Sci. 1995; 4: 1750-1757Crossref PubMed Scopus (71) Google Scholar, 6Shao Z. Arnold F.H. Curr. Opin. Struct. Biol. 1996; 6: 513-518Crossref PubMed Scopus (105) Google Scholar, 7Yano T. Oue S. Kagamiyama H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5511-5515Crossref PubMed Scopus (175) Google Scholar, 8Oue S. Okamoto A. Yano T. Kagamiyama H. J. Biol. Chem. 1999; 274: 2344-2349Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 9Mouratou B. Kasper P. Gehring H. Christen P. J. Biol. Chem. 1999; 274: 1320-1325Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar; for a review, see Ref. 6Shao Z. Arnold F.H. Curr. Opin. Struct. Biol. 1996; 6: 513-518Crossref PubMed Scopus (105) Google Scholar). Among the pyridoxal 5′-phosphate-dependent enzymes, aspartate aminotransferase (AspAT) 1The abbreviations used are:AspATaspartate aminotransferasePLPpyridoxal 5′-phosphateB6 enzymePLP (vitamin B6)-dependent enzymePMPpyridoxamine 5′-phosphate has been converted by multiple active-site mutations into anl-tyrosine aminotransferase (5Onuffer J.J. Kirsch J.F. Protein Sci. 1995; 4: 1750-1757Crossref PubMed Scopus (71) Google Scholar) and by directed molecular evolution into an l-branched-chain amino acid aminotransferase (7Yano T. Oue S. Kagamiyama H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5511-5515Crossref PubMed Scopus (175) Google Scholar, 8Oue S. Okamoto A. Yano T. Kagamiyama H. J. Biol. Chem. 1999; 274: 2344-2349Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Tyrosine phenol-lyase has been engineered by a double mutation to act as a dicarboxylic-acid β-lyase (an enzyme not found in nature) that degrades aspartate to pyruvate, ammonia, and formate (9Mouratou B. Kasper P. Gehring H. Christen P. J. Biol. Chem. 1999; 274: 1320-1325Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). However, as yet, no change in the reaction specificity of an enzyme has been reported, with the exception of the conversion of papain into a peptide-nitrile hydratase (10Dufour E. Storer A.C. Ménard R. Biochemistry. 1995; 34: 16382-16388Crossref PubMed Scopus (53) Google Scholar). A change in the reaction specificity may be claimed if a new catalytic activity not inherent in the wild-type enzyme is generated and the original activity of the wild-type enzyme is suppressed to a level significantly below that of the new activity. aspartate aminotransferase pyridoxal 5′-phosphate PLP (vitamin B6)-dependent enzyme pyridoxamine 5′-phosphate The pyridoxal 5′-phosphate (PLP)-dependent enzymes (B6 enzymes) catalyze numerous reactions in the metabolism of amino acids. The B6 enzymes are of multiple evolutionary origin and constitute a few families of homologous proteins of which the α-family is by far the largest (11Alexander F.W. Sandmeier E. Mehta P.K. Christen P. Eur. J. Biochem. 1994; 219: 953-960Crossref PubMed Scopus (346) Google Scholar). The enzyme members of the α-family, which includes AspAT, not only possess similar protein scaffolds, but most of them also share the first two steps of the reaction pathway (for a succinct introduction into PLP-dependent reaction pathways, see Ref. 12Metzler D.E. Biochemistry. Academic Press, Orlando, FL1977: 444-461Google Scholar). The amino group of the incoming substrate replaces the ε-amino group of the active-site lysine residue, the internal aldimine 1 (see Scheme FS1), thus being followed by the external aldimine intermediate2, which is then deprotonated at C-α to give the quinonoid intermediate 3. Only in the subsequent step do the reaction pathways of the different B6 enzymes diverge, leading to racemization, transamination, β- and γ-elimination and replacement. It seems therefore feasible to make the quinonoid intermediate3 in a given enzyme adopt an alternative reaction course by substituting few critical active-site residues. Aspartate aminotransferase is the most extensively studied B6 enzyme. The homodimeric enzyme (2 × 400 amino acid residues) catalyzes the reversible transfer of the amino group of aspartate or glutamate to the cognate oxo acids. A detailed mechanism of action has been derived from combined biochemical and crystallographic data (13Kirsch J.F. Eichele G. Ford G.C. Vincent M.G. Jansonius J.N. Gehring H. Christen P. J. Mol. Biol. 1984; 174: 497-525Crossref PubMed Scopus (425) Google Scholar). In a previous study, we have generatedl-aspartate β-decarboxylase activity in AspAT ofEscherichia coli by introducing a double active-site mutation (14Graber R. Kasper P. Malashkevich V.N. Sandmeier E. Berger P. Gehring H. Jansonius J.N. Christen P. Eur. J. Biochem. 1995; 232: 686-690Crossref PubMed Scopus (39) Google Scholar). AspAT Y225R/R386A β-decarboxylatedl-aspartate to l-alanine withk cat′ = 0.08 s−1, i.e.1330-fold faster than the wild-type enzyme. However, transaminase activity, despite a decrease by 3 orders of magnitude, still exceeded β-decarboxylase activity by a factor of 2.5. Here we searched for a third mutation that, if introduced into AspAT Y225R/R386A, would decrease further transaminase activity without affecting β-decarboxylase activity. The only mutation among many tested that brought about this effect was the replacement of the second active-site arginine residue, i.e. Arg292 (a residue of the adjacent subunit of the AspAT homodimer) with lysine. In the wild-type enzyme, Arg292 binds the distal carboxylate group of the substrate (Fig. 1). The single R292K mutation had been previously found to decrease transaminase activity to 0.2% of that of the wild-type enzyme (15Vacca R.A. Giannattasio S. Graber R. Sandmeier E. Marra E. Christen P. J. Biol. Chem. 1997; 272 (, and references cited therein): 21932-21937Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In the triple-mutant enzyme, β-decarboxylase activity indeed exceeded transaminase activity by a factor of 8. Oligonucleotide-directed mutagenesis of the wild-type aspC gene of E. coli inserted into the BS M13 vector (16Malcolm B.A. Kirsch J.F. Biochem. Biophys. Res. Commun. 1985; 132: 915-921Crossref PubMed Scopus (64) Google Scholar) was performed with the mutagenesis kit from Bio-Rad. The mutations were confirmed by determination of the nucleotide sequences. The mutated DNAs were expressed in the AspAT-deficientE. coli strain TY103 (17Yano T. Kuramitsu S. Tanase S. Morino Y. Hiromi K. Kagamiyama H. J. Biol. Chem. 1991; 266: 6079-6085Abstract Full Text PDF PubMed Google Scholar) with the expression vector pKDHE19 (18Kamitori S. Hirotsu K. Higuchi T. Kondo K. Inoue K. Kuramitsu S. Kagamiyama H. Higuchi Y. Yasuoka N. Kusunoki M. Matsuura Y. J. Biochem. ( Tokyo ). 1987; 101: 813-816Crossref PubMed Scopus (47) Google Scholar). Wild-type and mutant enzymes were purified with previously described chromatographic procedures. Fractions containing pure AspAT were pooled, concentrated, and reconstituted with coenzyme as described (19Vacca R.A. Christen P. Malashkevich V.N. Jansonius J.N. Sandmeier E. Eur. J. Biochem. 1995; 227: 481-487Crossref PubMed Scopus (17) Google Scholar). The concentration of the purified enzymes in the PLP form was determined spectrophotometrically at 280 nm using the molar absorption coefficient of the subunit, ε = 4.7 × 104m−1 cm−1 (20Kuramitsu S. Hiromi K. Hayashi H. Morino Y. Kagamiyama H. Biochemistry. 1990; 29: 5469-5476Crossref PubMed Scopus (169) Google Scholar). Kinetic parameters for aminotransferase activities of AspAT mutants and the wild-type enzyme were measured in a coupled assay with malate dehydrogenase containing 40 mml-aspartate plus 20 mm 2-oxoglutarate as substrates for the wild-type enzyme and 100 mml-aspartate plus 50 mm2-oxoglutarate for the mutant enzymes. The values ofk cat refer to subunit concentrations. TheK m′ values for l-aspartate and 2-oxoglutarate were measured at fixed concentrations of 2-oxoglutarate (50 mm) and l-aspartate (200 mm), respectively. For measuring the consumption and production of oxo acids, the enzymes (0.45 mm, subunit concentration) were incubated with 200 mml-aspartate and 8 mm oxalacetate in 250 mm 4-methylmorpholine (pH 7.5) at 25 °C. Samples (40 μl) were deproteinized at different time intervals with 1m perchloric acid (final concentration) and neutralized with potassium hydroxide. Oxalacetate and pyruvate were determined separately by consumption of NADH in the presence of malate dehydrogenase and lactate dehydrogenase, respectively. The β-decarboxylation of oxalacetate in the absence of enzyme (t½ = 60 min under the conditions detailed in the legend of Fig. 2 with 0.45 mm PLP added) was neglected in the calculations of k cat for the different enzyme variants. AspATs were incubated with amino acid and their cognate oxo acid in 250 mm 4-methylmorpholine (pH 7.5). High buffer concentrations are needed in the assays because CO2 is released in the β-decarboxylation reaction. The β-decarboxylase activity of the two mutant enzymes is sensitive to pH; a deviation by 0.5 from the optimum at pH 7.5 decreases the activity by 50% (data not shown). After addition of 0.5 μmol of l-valine as internal standard, 20-μl deproteinized samples of the reaction mixture were derivatized with 2-fluoro-2,4-dinitrophenyl-5-l-alanine amide (Marfey's reagent) and analyzed as described previously (15Vacca R.A. Giannattasio S. Graber R. Sandmeier E. Marra E. Christen P. J. Biol. Chem. 1997; 272 (, and references cited therein): 21932-21937Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar,21Kochhar S. Christen P. Eur. J. Biochem. 1988; 175: 433-438Crossref PubMed Scopus (26) Google Scholar). For the determination of the rates of desulfination ofl-cysteine sulfinate, the enzymes (0.45 mm, subunit concentration) were incubated with 100 mml-cysteinesulfinic acid and 50 mm2-oxoglutarate in 200 mm 4-methylmorpholine (pH 7.5) at 25 °C, and the production of alanine was measured as described above. To check which pathway of β-elimination the enzymes were following (see Scheme FS1), the same experiments were performed in the presence of 45 units/ml lactate dehydrogenase and 50 mmNADH to trap any pyruvate produced by hydrolysis of the ketimine intermediate 9 (see Scheme FS1). Transamination ofl-cysteine sulfinate was quantified by the increase in the concentration of l-glutamate produced by the reaction of the PMP form of the enzyme with oxoglutarate. Crystals of AspAT Y225R/R292K/R386A were grown with the hanging drop technique. A solution containing 15 mg/ml protein, 2 mm5′-phosphopyridoxyl l-aspartate (13Kirsch J.F. Eichele G. Ford G.C. Vincent M.G. Jansonius J.N. Gehring H. Christen P. J. Mol. Biol. 1984; 174: 497-525Crossref PubMed Scopus (425) Google Scholar), and 50 mm4-methylmorpholine (pH 7.5) was mixed 1:1 with reservoir solution containing 1.98 m ammonium sulfate, 2% (w/v) polyethylene glycol (M r 400), and 200 mm4-methylmorpholine (pH 7.5). Crystals grew to a maximum size of 0.2 × 0.4 × 1.5 mm in ∼4 weeks. Nucleation and crystal growth proved more problematic than in the case of wild-type AspAT, probably indicating a less stable conformation of the protein. As found before for crystals of wild-type and mutant E. coli AspATs (14Graber R. Kasper P. Malashkevich V.N. Sandmeier E. Berger P. Gehring H. Jansonius J.N. Christen P. Eur. J. Biochem. 1995; 232: 686-690Crossref PubMed Scopus (39) Google Scholar, 22Jäger J. Moser M. Sauder U. Jansonius J.N. J. Mol. Biol. 1994; 239: 285-305Crossref PubMed Scopus (175) Google Scholar), the crystals belong to space group P21 with unit cell dimensions a = 88.03 Å, b = 80.32 Å, c = 87.82 Å, and β = 119.85 °. Diffraction data were collected to a resolution of 2.16 Å, using a MARresearch imaging plate mounted on a modified Elliott GX20 rotating copper anode generator. Raw data were processed with MOSFLM (23Leslie A.G.V. MOSFLM Users Guide. Medical Research Council-Laboratory of Molecular Biology, Cambridge1994Google Scholar). Images were scaled with SCALA and AGROVATA and reduced to structure factors with TRUNCATE from the CCP4 Program Suite (24CCP4 Program Suite: Collaborative Computing Project No. 4 Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19769) Google Scholar). A total of 170,054 measured reflections were merged together withR sym = 0.082 to give rise to the data set of 50,068 independent reflections, which is 87.7% complete to 2.16-Å resolution. The structure of the 5′-phosphopyridoxyl aspartate complex of AspAT Y225R/R292K/R386A was solved by molecular replacement with the program AMORE (24CCP4 Program Suite: Collaborative Computing Project No. 4 Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19769) Google Scholar), using the refined structure of the open form of the wild-type enzyme (22Jäger J. Moser M. Sauder U. Jansonius J.N. J. Mol. Biol. 1994; 239: 285-305Crossref PubMed Scopus (175) Google Scholar) as search model, and refined with TNT (25Tronrud D.E. Ten Eyck L.F. Matthews B.W. Acta Crystallogr. Sect. A. 1987; 43: 489-501Crossref Scopus (874) Google Scholar) tor = 0.19. The root mean square deviations of the bond lengths, bond angles, and planes from the ideal values were 0.011 Å, 1.35°, and 0.01 Å, respectively. The simulations of the quinonoid intermediates 3 (see Scheme FS1) were performed as described previously (14Graber R. Kasper P. Malashkevich V.N. Sandmeier E. Berger P. Gehring H. Jansonius J.N. Christen P. Eur. J. Biochem. 1995; 232: 686-690Crossref PubMed Scopus (39) Google Scholar). The cell multipole method (26Greengard L. Rokhlin V.I. J. Comp. Physiol. 1987; 73: 325-330Crossref Scopus (3470) Google Scholar) was used instead of a cutoff for the nonbonded interactions. The modeled structure of the triple-mutant enzyme was obtained by the replacement of Arg292 with a lysine residue in the double-mutant crystal structure. The systems were relaxed by 4000 steps of energy minimization. The amino acid residues and water molecules beyond a distance of 11 Å from the coenzyme-substrate adduct were kept fixed during the following 100 ps of simulation at 300 K. The introduction of the additional mutation R292K into AspAT Y225R/R386A did not affect its l-aspartate β-decarboxylase activity (Fig. 2); the k catand K m′ values of the triple- and double-mutant enzymes are the same (Table I). Two different pathways to produce l-alanine froml-aspartate are possible, i.e. direct β-decarboxylation of l-aspartate (7 →8 → 1 in SchemeFS1) or β-decarboxylation coupled with transamination (7 → 9 → 5), producing pyruvate, which, by transamination, may be converted tol-alanine. To determine the partition ratio of the two pathways, the consumption of oxalacetate (consumed in the reaction with the PMP form of the enzyme 5 to produce the PLP form1 and l-aspartate) and the production of pyruvate in the presence of l-aspartate and oxalacetate (conditions as described in the legend to Fig. 2) were followed in parallel with the β-decarboxylation of l-aspartate (TableII). Both AspAT Y225R/R386A and AspAT Y225R/R292K/R386A produced pyruvate with a k catof only 0.01 s−1, corresponding to a partition ratio (7 → 8 versus 7 →9) of 8. In the wild-type enzyme, production of pyruvate by far exceeded that of l-alanine. Probably, most of thel-alanine produced by the wild-type enzyme was formed by transamination of pyruvate.Table IKinetic parameters for β-decarboxylase and transaminase activities of wild-type and mutant AspATsAspATβ-Decarboxylation k catTransamination k catK m(Asp)′K m(OG)′aOG, 2-oxoglutarate; ND, not determined; BD, below detection limit.s−1s−1mmmmY225R/R292K/R386A0.080.018.80.8Y225R/R292E/R386A0.0055 × 10−5NDNDY225R/R292V/R386A 8 × 10−40.0516.80.36Y225R/R292Y/R386ABD0.02520014Y225R/R386A0.080.198.30.8R292K/R386A7.5 × 10−40.094140.25Y225RBD0.450.83bFrom Ref. 27.0.36bFrom Ref. 27.R292K1.8 × 10−30.5cFrom Ref. 15.14cFrom Ref. 15.2.7cFrom Ref. 15.R386ABD0.33155Wild-type6 × 10−5 dMost of the l-alanine produced in the presence of the wild-type enzyme is probably due to transamination of pyruvate formed by β-decarboxylation of oxalacetate.1801.20.2The β-decarboxylase assay was carried out in 200 mml-aspartate plus 8 mm oxalacetate in 250 mm 4-methylmorpholine (pH 7.5). Transaminasc activity was measured in the presence of 20 mml-aspartate plus 20 mm 2-oxoglutarate in 50 mm4-methylmorpholine (pH 7.5) for the wild-type enzyme and 100 mml-aspartate plus 50 mm2-oxoglutarate in 100 mm 4-methylmorpholine (pH 7.5) for the mutant enzymes. Because the transaminase activity of AspAT Y225R/R292E/R386A was too low to be analyzed with a coupled assay, the transformation of the PLP form of the enzyme into the PMP form upon addition of 100 mml-aspartate was followed spectrophotometrically instead. The disappearance of the PLP form and the appearance of the PMP form of the enzyme were recorded at 360 and 330 nm, respectively. All reactions were run at 25 °C. One double-mutant enzyme, AspAT Y225R/R292K, was not expressible inE. coli TY103. In the cell crude extract, no soluble enzyme could be detected on SDS-polyacrylamide gel after silver staining.a OG, 2-oxoglutarate; ND, not determined; BD, below detection limit.b From Ref. 27Inoue K. Kuramitsu S. Okamoto A. Hirotsu K. Higuchi T. Morino Y. Kagamiyama H. J. Biochem. ( Tokyo ). 1991; 109: 570-576Crossref PubMed Scopus (39) Google Scholar.c From Ref. 15Vacca R.A. Giannattasio S. Graber R. Sandmeier E. Marra E. Christen P. J. Biol. Chem. 1997; 272 (, and references cited therein): 21932-21937Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar.d Most of the l-alanine produced in the presence of the wild-type enzyme is probably due to transamination of pyruvate formed by β-decarboxylation of oxalacetate. Open table in a new tab Table IIComparison of kcat values for β-decarboxylation and transamination with those of β-desulfinationAspATkcatl-Aspartatel-Cysteine sulfinateβ-DecarboxylationaValues are taken from Table I.TransaminationaValues are taken from Table I.Pyruvate productionβ-DesulfinationTransaminations−1Y225R/R292K/R386A0.080.010.010.020.002Y225R/R386A0.080.190.010.1650.024Wild-type6 × 10−51800.00160.05512bFrom Ref. 28.Desulfination activity was determined by incubating 0.45 mmenzyme (subunit concentration) with 100 mml-cysteine sulfinate and 50 mm 2-oxoglutarate in 200 mm 4-methylmorpholine (pH 7.5) at 25 °C. Transamination of l-cysteine sulfinate was measured by the amount of l-glutamate produced in the regeneration of the PLP form from the PMP form of the enzyme (for details, see “Experimental Procedures”).a Values are taken from Table I.b From Ref. 28Furumo N.C. Kirsch J.F. Arch. Biochem. Biophys. 1995; 319: 49-54Crossref PubMed Scopus (11) Google Scholar. Open table in a new tab The β-decarboxylase assay was carried out in 200 mml-aspartate plus 8 mm oxalacetate in 250 mm 4-methylmorpholine (pH 7.5). Transaminasc activity was measured in the presence of 20 mml-aspartate plus 20 mm 2-oxoglutarate in 50 mm4-methylmorpholine (pH 7.5) for the wild-type enzyme and 100 mml-aspartate plus 50 mm2-oxoglutarate in 100 mm 4-methylmorpholine (pH 7.5) for the mutant enzymes. Because the transaminase activity of AspAT Y225R/R292E/R386A was too low to be analyzed with a coupled assay, the transformation of the PLP form of the enzyme into the PMP form upon addition of 100 mml-aspartate was followed spectrophotometrically instead. The disappearance of the PLP form and the appearance of the PMP form of the enzyme were recorded at 360 and 330 nm, respectively. All reactions were run at 25 °C. One double-mutant enzyme, AspAT Y225R/R292K, was not expressible inE. coli TY103. In the cell crude extract, no soluble enzyme could be detected on SDS-polyacrylamide gel after silver staining. Desulfination activity was determined by incubating 0.45 mmenzyme (subunit concentration) with 100 mml-cysteine sulfinate and 50 mm 2-oxoglutarate in 200 mm 4-methylmorpholine (pH 7.5) at 25 °C. Transamination of l-cysteine sulfinate was measured by the amount of l-glutamate produced in the regeneration of the PLP form from the PMP form of the enzyme (for details, see “Experimental Procedures”). Replacement of Arg292 in AspAT Y225R/R386A with a glutamate or valine residue led to 16- and 100-fold decreases ink cat for β-decarboxylation, respectively (Table I). Arg292 was substituted with glutamate to introduce a negative charge that might destabilize the β-carboxylate group (29Leussing D.L. Raghavan N.V. J. Am. Chem. Soc. 1980; 102: 5635-5643Crossref Scopus (24) Google Scholar,30Hurley J.H. Remington S.J. J. Am. Chem. Soc. 1992; 114: 4769-4773Crossref Scopus (11) Google Scholar) and thus enhance β-decarboxylation. If the ratio of β-decarboxylase to transaminase activity is considered rather than the absolute k cat value, AspAT Y225R/R292E/R386A is indeed a more specific l-aspartate β-decarboxylase than its counterpart with the R292K substitution, its β-decarboxylase activity being 100 times higher than its transaminase activity (TableI). Replacement of Arg292 with tyrosine eliminated the positive charge, whereas the phenolic hydroxy group could still form a hydrogen bond with the β-carboxylate group of the substrate and thus maintain the binding function. However, AspAT Y225R/R292Y/R386A proved to have very low affinity for the substrates and no measurable β-decarboxylase activity. The replacement of Arg292 with lysine, glutamate, valine, or tyrosine in AspAT Y225R/R386A led to a further marked decrease in k cat for transamination (Table I). However, only with the R292K substitution as the third mutation was β-decarboxylase activity maintained. TheK m values for dicarboxylic substrates of the single-, double-, and triple-mutant enzymes are invariably higher than those of the wild-type enzyme, with the exception of the single Y225R mutation, which has been reported to decrease the K mvalues for C4 and C5 dicarboxylic substrates (14Graber R. Kasper P. Malashkevich V.N. Sandmeier E. Berger P. Gehring H. Jansonius J.N. Christen P. Eur. J. Biochem. 1995; 232: 686-690Crossref PubMed Scopus (39) Google Scholar, 27Inoue K. Kuramitsu S. Okamoto A. Hirotsu K. Higuchi T. Morino Y. Kagamiyama H. J. Biochem. ( Tokyo ). 1991; 109: 570-576Crossref PubMed Scopus (39) Google Scholar, 31Goldberg J.M. Swanson R.V. Goodman H.S. Kirsch J.F. Biochemistry. 1991; 30: 305-312Crossref PubMed Scopus (82) Google Scholar, 32Goldberg J.M. Kirsch J.F. Biochemistry. 1996; 35: 5280-5291Crossref PubMed Scopus (61) Google Scholar). In AspAT Y225R/R292K/R386A in particular, theK m values for l-aspartate and 2-oxoglutarate are, as in the Y225R/R386A double-mutant enzyme, seven and four times higher, respectively, than in the wild-type enzyme. AspAT Y225R/R292E/R386A was also tested for activity towardl-lysine, l-arginine, andl-ornithine. A very slow transamination reaction ofl-lysine with an initial rate of 0.001 s−1could be detected. Such an effect of a negative charge at position 292 has been reported previously (33Cronin C.N. Kirsch J.F. Biochemistry. 1988; 27: 4572-4579Crossref PubMed Scopus (109) Google Scholar, 34Hwang J.K. Warshel A. Nature. 1988; 334: 270-272Crossref PubMed Scopus (85) Google Scholar, 35Almo S.C. Smith D.L. Danishefsky A.T. Ringe D. Protein Eng. 1994; 7: 405-412Crossref PubMed Scopus (35) Google Scholar). Under the same conditions, no reaction of l-lysine with the wild-type enzyme was observed. None of the mutant enzymes showed any measurable reaction other than transamination toward d/l-glutamate,d-aspartate, l-tyrosine, orl-serine and their cognate oxo acids. Wild-type AspAT catalyzed the transamination of l-cysteine sulfinate at a very high rate. As a side reaction, elimination of sulfinate produced l-alanine (Table II). Both AspAT Y225R/R386A and AspAT Y225R/R292K/R386A showed a reaction specificity that was inverse to that of the wild-type enzyme, desulfination ofl-cysteine sulfinate being by an order of magnitude faster than its transamination reaction. The double mutation increased desulfination activity 3-fold and decreased transaminase activity toward l-cysteine sulfinate by 4 orders of magnitude. The introduction of the third mutation (R292K) reduced both desulfination and the transamination activity of the double-mutant enzyme by an order of magnitude. Lactate dehydrogenase plus NADH had no effect on k cat of desulfination by the wild-type and mutant AspATs, indicating that l-alanine is produced by direct desulfination of l-cysteine sulfinate rather than through formation of pyruvate followed by transamination. The 5′-phosphopyridoxyl aspartate complex of the triple-mutant enzyme (Y225R/R292K/R386A) was found in the open conformation (Fig. 1). In the wild-type enzyme, binding of dicarboxylic substrates or inhibitors induces the closed conformation of the enzyme, in which water molecules are excluded from the vicinity of the Schiff base (13Kirsch J.F. Eichele G. Ford G.C. Vincent M.G. Jansonius J.N. Gehring H. Christen P. J. Mol. Biol. 1984; 174: 497-525Crossref PubMed Scopus (425) Google Scholar). The open conformation of the triple-mutant enzyme allows water molecules to enter the active site in the presence of the substrate analog. Lys258, which is responsible in the wild-type enzyme (13Kirsch J.F. Eichele G. Ford G.C. Vincent M.G. Jansonius J.N. Gehring H. Christen P. J. Mol. Biol. 1984; 174: 497-525Crossref PubMed Scopus (425) Google Scholar) for the deprotonation at C-α and reprotonation at C-4′ of the coenzyme (Scheme FS1), has moved away from its position near these atoms, where a water molecule is now found. The amino group of Lys258 is within hydrogen-bonding distance to Arg225. Lys292 does not interact with the distal carboxylate group of the aspartate moiety; it forms a hydrogen bond with Ser296 instead, whereas a water molecule occupies its original position. The electron density of the aspartate moiety is highly disordered due to the lack of Arg292 and Arg386, which are key residues for substrate binding in the wild-type enzyme. Nevertheless, the coenzyme-substrate adduct maintains a conformation that allows elimination of the proton at C-α, the C-α–H bond together with the imine nitrogen staying in a plane orthogonal to the plane defined by the coenzyme ring (13Kirsch J.F. Eichele G. Ford G.C. Vincent M.G. Jansonius J.N. Gehring H. Christen P. J. Mol. Biol. 1984; 174: 497-525Crossref PubMed Scopus (425) Google Scholar, 36Dunathan H.C. Proc. Natl. Acad. Sci. U. S. A. 1966; 55: 712-716Crossref PubMed Scopus (343) Google Scholar). In the simulation of the external aldimine intermediate based on the crystal structure of AspAT Y225R/R292K/R386A (Fig. 1), Lys258 did not displace the intervening water molecule and approach C-α. This situation most probably is due to a crystal artifact as it would correspond to a catalytically inactive enzyme. The dynamics calculations for the quinonoid intermediate of the triple-mutant enzyme were therefore based on the crystal structure of AspAT Y225R/R386A (14Graber R. Kasper P. Malashkevich V.N. Sandmeier E. Berger P. Gehring H. Jansonius J.N. Christen P. Eur. J. Biochem. 1995; 232: 686-690Crossref PubMed Scopus (39) Google Scholar), in which Arg292 had been replaced with a lysine residue. In this case, Lys258 stayed close enough to C-α and C-4′ for acting as the acid-base group in the tautomerization from aldimine 2to ketimine 4. In the molecular dynamics simulation of the quinonoid intermediate of AspAT Y225R/R292K/R386A, as in those of AspAT Y225R/R386A, a hydrogen bond between Arg225 and the imine nitrogen atom is formed (Fig. 3). During the simulation, this hydrogen bond exists only 5% of the time in AspAT Y225R, whereas it is present 35% of the time in the double- and triple-mutant enzymes. In all AspATs containing the Y225R mutation, the proximity of the positively charged guanidinium group repulses the protonated Lys258. Its longer distance from C-4′ of the coenzyme hinders reprotonation of that atom and might underlie the decrease in transaminase activity. In the simulated structures of the quinonoid intermediate of the wild-type enzyme, Lys258remains positioned above the imine nitrogen at almost equal distance from C-α and C-4′. The triple mutation Y225R/R292K/R386A brings about a switch in the reaction specificity of E. coli AspAT. The conjoint R386A and Y225R substitutions enhance the very low l-aspartate β-decarboxylase activity of the wild-type enzyme and decrease transaminase activity. Measurements of the activity of the pertinent single- and double-mutant enzymes other than Y225R/R386A confirmed that the increase in β-decarboxylase activity strictly depends on the combined effects of the R386A and Y225R substitutions (Table I). This double mutation amounts to a shift of an arginine residue from position 386 to position 225. The third mutation, replacement of Arg292with lysine, selectively lowers transaminase activity to one-eighth of β-decarboxylase activity. Together, the three mutations increase the ratio of β-decarboxylase to transaminase activity >25 million-fold. Molecular dynamics simulations of the wild-type and triple-mutant enzymes based on crystal structures suggested, as previously for the double-mutant enzyme, that Arg225 makes, in addition to the hydrogen bond with O-3′ of the coenzyme, a second hydrogen bond with the imine nitrogen of the quinonoid intermediate. In the wild-type enzyme, the imine nitrogen is not engaged in a hydrogen bond, whereas O-3′ forms a hydrogen bond with Tyr225. The single Y225R mutation is apparently not sufficient to give rise to the formation of a hydrogen bond from Arg225 to the imine nitrogen atom (14Graber R. Kasper P. Malashkevich V.N. Sandmeier E. Berger P. Gehring H. Jansonius J.N. Christen P. Eur. J. Biochem. 1995; 232: 686-690Crossref PubMed Scopus (39) Google Scholar). The mutation has been found to decrease the pK a of the internal aldimine from 6.8 in the wild-type enzyme to 6.2 due to a strong hydrogen bond of Arg225 with O-3′ (27Inoue K. Kuramitsu S. Okamoto A. Hirotsu K. Higuchi T. Morino Y. Kagamiyama H. J. Biochem. ( Tokyo ). 1991; 109: 570-576Crossref PubMed Scopus (39) Google Scholar), which pulls electrons out of the π-system extending from the pyridine ring of PLP to the aldimine bond. The imine nitrogen might thus become a too weak nucleophile to accept a hydrogen bond from Arg225. The replacement of Arg386 by an alanine residue has been reported previously to disrupt the hydrogen-bonding network Arg386 → Asn194 → O-3′ (37Yano T. Mizuni T. Kagamiyama H. Biochemistry. 1993; 32: 1810-1815Crossref PubMed Scopus (46) Google Scholar) and thus to allow the electrons of O-3′ to flow into the π-system. As a consequence, the imine nitrogen in the double-mutant enzyme might engage in a hydrogen bond with Arg225. A similar effect of the Y225R/R386A mutations might be operative in the quinonoid intermediate (14Graber R. Kasper P. Malashkevich V.N. Sandmeier E. Berger P. Gehring H. Jansonius J.N. Christen P. Eur. J. Biochem. 1995; 232: 686-690Crossref PubMed Scopus (39) Google Scholar). The O-3′–Arg225 and imine nitrogen–Arg225hydrogen bonds may be assumed to reinforce the electron sink capacity of the π-system of imine and the cofactor pyridine ring to such an extent that, even after deprotonation at C-α, it remains effective enough to stabilize carbanion 6 (Scheme FS1), produced by β-decarboxylation. Other factors that might favor β-decarboxylation, such as the angle of the C-β–C-γ bond relative to the coenzyme-imine π-system (36Dunathan H.C. Proc. Natl. Acad. Sci. U. S. A. 1966; 55: 712-716Crossref PubMed Scopus (343) Google Scholar) and negative electrostatic potentials around the β-carboxylate group that might promote electron delocalization (38Gallagher T. Snell E.E. Hackert M.L. J. Biol. Chem. 1989; 264: 12737-12743Abstract Full Text PDF PubMed Google Scholar), could not be verified; neither the bond angles nor the electrostatic potentials in AspAT Y225R/R292K/R386A were significantly different from those in the wild-type enzyme. The decrease in transaminase activity observed in both AspAT Y225R/R386A and AspAT Y225R/R292K/R386A might be due to the repulsion between Lys258 and Arg225 impairing the reprotonation of the quinonoid intermediate 3 at C-4′. A possible explanation for the further decrease brought about by the third mutation (R292K) is provided by the recent crystallographic analysis of three reaction intermediates of the wild-type enzyme7 that has shown a water molecule to be positioned near C-α in the ketimine intermediate 4; this water molecule is supposed to effect the hydrolysis of the ketimine. 2V. N. Malashkevich and J. N. Jansonius, unpublished data. Its nucleophilicity might be enhanced by a hydrogen-bonding network comprising Tyr70, Lys258, and Gly38. The effect of these interactions is maximal if the active site is in the closed conformation because the rotation of the small domain brings Gly38 into a position where it can take part in this network. The wild-type enzyme assumes the closed conformation on binding a dicarboxylic amino or oxo acid to the two active-site arginine residues, Arg292 and Arg386. By interacting with the substrate, the two arginines are pulled toward each other, inducing the domain movement (13Kirsch J.F. Eichele G. Ford G.C. Vincent M.G. Jansonius J.N. Gehring H. Christen P. J. Mol. Biol. 1984; 174: 497-525Crossref PubMed Scopus (425) Google Scholar, 22Jäger J. Moser M. Sauder U. Jansonius J.N. J. Mol. Biol. 1994; 239: 285-305Crossref PubMed Scopus (175) Google Scholar, 39Picot D. Sandmeier E. Thaller C. Vincent M.G. Christen P. Jansonius J.N. Eur. J. Biochem. 1991; 196: 329-341Crossref PubMed Scopus (53) Google Scholar). In AspAT Y225R/R292K/R386A, the mutation of both arginine residues prevents the enzyme from adopting a closed conformation (Fig. 1), with the consequence that the water molecule might not be reactive enough to attack C-α. In AspAT Y225R/R386A, with only one substrate-binding arginine missing, the syncatalytic closure of the active-site cleft is partially retained (14Graber R. Kasper P. Malashkevich V.N. Sandmeier E. Berger P. Gehring H. Jansonius J.N. Christen P. Eur. J. Biochem. 1995; 232: 686-690Crossref PubMed Scopus (39) Google Scholar). The importance of the domain movement for the reactivity of the catalytic water molecule in the hydrolysis of the ketimine intermediate might also explain the reaction pathway of both β-decarboxylation and β-desulfination. The amino acid l-cysteine sulfinate is a dianion like aspartate and is a physiologic substrate for AspAT (40Parsons B. Rainbow T.C. Brain Res. 1984; 294: 193-197Crossref PubMed Scopus (13) Google Scholar,41Weinstein C.L. Haschemeyer R.H. Griffith O.W. J. Biol. Chem. 1988; 263: 16568-16579Abstract Full Text PDF PubMed Google Scholar). The reaction specificity of both AspAT Y225R/R386A and AspAT Y225R/R292K/R386A toward l-cysteine sulfinate is inverse to that of the wild-type enzyme. The mutant enzymes desulfinate this substrate faster than they undergo the transamination reaction with it. Nevertheless, similar to the wild-type enzyme and in analogy to the β-decarboxylation reaction with aspartate, they preferentially reprotonate carbanion 7 (Scheme FS1) at C-α rather than C-4′ and produce l-alanine (7 → 8 →1) rather than pyruvate (7 → 9 →5). Conceivably, upon loss of the negatively charged β-substituent, the active site assumes the open conformation. Thus, the frequency of ketimine hydrolysis is decreased, and the partition ratio is shifted in favor of reprotonation at C-α, resulting in the production of l-alanine. The starting point of this work was a study of the molecular evolution of B6 enzymes (11Alexander F.W. Sandmeier E. Mehta P.K. Christen P. Eur. J. Biochem. 1994; 219: 953-960Crossref PubMed Scopus (346) Google Scholar). Within a given family, in particular in the large α-family, a clear temporal sequence of different phases in the functional specialization is evident. The common ancestor enzyme, apparently an unspecific all-rounder catalyst, first diverged into reaction-specific protoenzymes, which then diverged further and acquired substrate specificity. The last phase for most B6enzymes was the neutral evolution concomitant with speciation. 3Mehta, P. K., and Christen, P. (2000) Adv. Enzymol. Relat. Areas Mol. Biol., in press. The conjoint substitution of three active-site residues that converted AspAT into anl-aspartate β-decarboxylase seems to simulate the processes that, in the first phase of molecular evolution, might have led to reaction-specific B6 enzymes by accelerating the specific reaction and suppressing potential side reactions. To the best of our knowledge, such a clear change in reaction specificity with a remarkably high new activity (k cat = 0.08 s−1) has as yet only been reported for papain that was converted into a peptide-nitrile hydratase by a single amino acid substitution at the active site (10Dufour E. Storer A.C. Ménard R. Biochemistry. 1995; 34: 16382-16388Crossref PubMed Scopus (53) Google Scholar). Two features of the procedure used in this study for changing the reaction specificity might be generally applicable to B6enzymes and perhaps certain other enzymes as well. 1) The double mutation Y225R/R386A shifts an arginine residue from its wild-type position to another position in its immediate vicinity. Such charge-shifting double mutations may be expected not to disturb greatly the topochemistry of the active site, but to alter the electron repartition at the reaction center. 2) Arginine residues are the preferred binding groups for anionic substrates in enzymes (15Vacca R.A. Giannattasio S. Graber R. Sandmeier E. Marra E. Christen P. J. Biol. Chem. 1997; 272 (, and references cited therein): 21932-21937Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Their conservative substitution by lysine, which is slightly shorter and engages in fewer hydrogen bonds, may be expected to change the mode of binding of the substrate.