Portal de Periódicos da CAPES

Mutation of Tyrosine 332 to Phenylalanine Converts Dopa Decarboxylase into a Decarboxylation-dependent Oxidative Deaminase

2002; Elsevier BV; Volume: 277; Issue: 39 Linguagem: Inglês

10.1074/jbc.m204867200

1083-351X

Mariarita Bertoldi, Marco Gonsalvi, Roberto Contestabile, Carla Borri Voltattorni,

Biochemical and biochemical processes

A flexible loop (residues 328–339), presumably covering the active site upon substrate binding, has been revealed in 3,4-dihydroxyphenylalanine decarboxylase by means of kinetic and structural studies. The function of tyrosine 332 has been investigated by substituting it with phenylalanine. Y332F displays coenzyme content and spectroscopic features identical to those of the wild type. Unlike wild type, during reactions withl-aromatic amino acids under both aerobic and anaerobic conditions, Y332F does not catalyze the formation of aromatic amines. However, analysis of the products shows that in aerobiosis,l-aromatic amino acids are converted into the corresponding aromatic aldehydes, ammonia, and CO2 with concomitant O2 consumption. Therefore, substitution of Tyr-332 with phenylalanine results in the suppression of the original activity and in the generation of a decarboxylation-dependent oxidative deaminase activity. In anaerobiosis, Y332F catalyzes exclusively a decarboxylation-dependent transamination ofl-aromatic amino acids. A role of Tyr-332 in the Cα protonation step that catalyzes the formation of physiological products has been proposed. Furthermore, Y332F catalyzes oxidative deamination of aromatic amines and half-transamination of d-aromatic amino acids with k cat values comparable with those of the wild type. However, for all the mutant-catalyzed reactions, an increase in K m values is observed, suggesting that Y → F replacement also affects substrate binding. A flexible loop (residues 328–339), presumably covering the active site upon substrate binding, has been revealed in 3,4-dihydroxyphenylalanine decarboxylase by means of kinetic and structural studies. The function of tyrosine 332 has been investigated by substituting it with phenylalanine. Y332F displays coenzyme content and spectroscopic features identical to those of the wild type. Unlike wild type, during reactions withl-aromatic amino acids under both aerobic and anaerobic conditions, Y332F does not catalyze the formation of aromatic amines. However, analysis of the products shows that in aerobiosis,l-aromatic amino acids are converted into the corresponding aromatic aldehydes, ammonia, and CO2 with concomitant O2 consumption. Therefore, substitution of Tyr-332 with phenylalanine results in the suppression of the original activity and in the generation of a decarboxylation-dependent oxidative deaminase activity. In anaerobiosis, Y332F catalyzes exclusively a decarboxylation-dependent transamination ofl-aromatic amino acids. A role of Tyr-332 in the Cα protonation step that catalyzes the formation of physiological products has been proposed. Furthermore, Y332F catalyzes oxidative deamination of aromatic amines and half-transamination of d-aromatic amino acids with k cat values comparable with those of the wild type. However, for all the mutant-catalyzed reactions, an increase in K m values is observed, suggesting that Y → F replacement also affects substrate binding. 3,4-dihydroxyphenylalanine Dopa decarboxylase pyridoxal 5′-phosphate pyridoxamine 5′-phosphate 5-hydroxytryptophan 5-hydroxytryptamine (serotonin) high pressure liquid chromatography Dopa1 decarboxylase (DDC; EC 4.1.1.28) is a homodimeric pyridoxal 5′-phosphate (PLP) enzyme that catalyzes as the main reaction the decarboxylation ofl-aromatic amino acids into the corresponding aromatic amines shown in Reaction 1. L­aromatic amino acid→aromatic amine+CO2 REACTION1Side reactions with turnover times measured in minutes are also catalyzed by the enzyme. In particular, DDC exhibits half-transaminase activity toward d-aromatic amino acids (1Bertoldi M. Frigeri P. Paci M. Borri Voltattorni C. J. Biol. Chem. 1999; 274: 5514-5521Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) and oxidative deaminase activity toward aromatic amines (2Bertoldi M. Moore P.S. Maras B. Dominici P. Borri Voltattorni C. J. Biol. Chem. 1996; 271: 23954-23959Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 3Bertoldi M. Dominici P. Moore P.S. Maras B. Borri Voltattorni C. Biochemistry. 1998; 37: 6552-6561Crossref PubMed Scopus (40) Google Scholar), as shown in Reactions 2 and 3. D­aromatic amino acid+PLP→aromatic ketoacid +pyridoxamine5′­phosphate(PMP) REACTION2 Aromatic amine+1/2O2→aromatic aldehyde+NH3 REACTION3Studies on the effect exerted by O2 on reaction specificity of the enzyme have shown that under anaerobic conditions, Reaction 1 takes place with a k cat value approximately half that occurring in the presence of O2 and is accompanied by a decarboxylation-dependent transamination (4Bertoldi M. Borri Voltattorni C. Biochem. J. 2000; 352: 533-538Crossref PubMed Scopus (38) Google Scholar), and Reaction 2 occurs at the same extent either in the presence or absence of O2 (4Bertoldi M. Borri Voltattorni C. Biochem. J. 2000; 352: 533-538Crossref PubMed Scopus (38) Google Scholar). Reaction 3 does not occur in anaerobiosis and is replaced by half-transamination (1Bertoldi M. Frigeri P. Paci M. Borri Voltattorni C. J. Biol. Chem. 1999; 274: 5514-5521Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 5Bertoldi M. Borri Voltattorni C. Protein Sci. 2001; 10: 1178-1186Crossref PubMed Scopus (15) Google Scholar). Tancini et al. (6Tancini B. Dominici P. Simmaco M. Schininà M.E. Barra D. Borri Voltattorni C. Arch. Biochem. Biophys. 1988; 260: 569-576Crossref PubMed Scopus (21) Google Scholar) reported the presence of an exposed and flexible region in the native pig kidney DDC molecule susceptible to tryptic digestion by which two fragments cleaved at the Lys-334–His-335 bond were formed. Although the nicked enzymatic species does not exhibit either decarboxylase or oxidative deamination activities, it retains a large percentage of the native transaminase activity toward d-aromatic amino acids and displays a slow transaminase activity toward aromatic amines (1Bertoldi M. Frigeri P. Paci M. Borri Voltattorni C. J. Biol. Chem. 1999; 274: 5514-5521Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Steady-state kinetic studies of native and nicked enzymatic species together with protection experiments against limited proteolysis of DDC by various substrates have suggested that ligand conformational changes occur at or near the tryptic cleavage region (1Bertoldi M. Frigeri P. Paci M. Borri Voltattorni C. J. Biol. Chem. 1999; 274: 5514-5521Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The finding that recombinant rat liver DDC lacking this loop is incompetent for decarboxylation supports this view (7Ishii S. Hayashi H. Okamoto A. Kagamiyama H. Protein Sci. 1998; 7: 1802-1810Crossref PubMed Scopus (27) Google Scholar). The spatial structure of ligand-free DDC and its complex with the anti-Parkinson drug carbiDopa has been recently solved, but the flexible loop between residues 328 and 339 is invisible in the electron density map (8Burkhard P. Dominici P. Borri Voltattorni C. Jansonius J.N. Malashkevich V.N. Nat. Struct. Biol. 2001; 8: 963-967Crossref PubMed Scopus (184) Google Scholar). A model based on the coordinates of the enzyme with the flexible loop in its hypothetical open form was built. Although in this modeled structure the loop is located at the dimer interface, far away from the active site, it is expected to extend toward the active site of the other monomer in a closed conformation upon substrate binding. Such loop closure could be an essential step in the catalytic mechanism of the enzyme, and it is also reasonable to suppose that some loop residues could even take part in the catalytic mechanism. Fig.1 shows a tentative model of the flexible loop in a position to gate access to the active site in the DDC-carbiDopa complex. The tyrosyl residue, Tyr-332, in the model is found 3.86 Å from the α-carbon of the ligand. This residue is of particular interest as it is conserved in PLP-dependent group II α-decarboxylases (9Ishii S. Mizuguchi H. Nishino J. Hayashi H. Kagamiyama H. J. Biochem. 1996; 120: 369-376Crossref PubMed Scopus (41) Google Scholar). Site-directed mutagenesis has been used to investigate the role of Tyr-332 in the mobile loop of DDC. l-Dopa, l- andd-5-hydroxytryptophan (5-HTP), 5-hydroxytryptamine (5-HT, serotonin), dopamine, PLP, pyridoxamine 5′-phosphate (PMP), NADH, bovine liver l-glutamate dehydrogenase, horse liver alcohol dehydrogenase, and Hepes were Sigma products. The liquid chromatography solvents (HPLC grade) were from Labscan. Ingredients for bacterial growth were from Difco. Oligonucleotides were from Invitrogen. PCR amplifications were performed using the Expand high fidelity PCR system commercialized by Stratagene. The restriction enzymes used for cloning were from Biolabs. d,l-[1-14C]Dopa (55 mCi/mmol) was a product of ICN Pharmaceuticals. All other chemicals were of the highest purity available. Site-directed mutagenesis was performed by overlap extension PCR (10Higuchi R. Innis M.A. Gelfand D.H. Snisky J.J. White T.J. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA1990: 177-183Google Scholar). This method uses four oligonucleotide primers in three separate PCR reactions to introduce a mutation into the target DNA sequence. Two separate PCR reactions are run, one using primers 1 and 3 to amplify a portion of the target sequence and the other using primers 2 and 4 to amplify the other portion. A short section of DNA has identical sequence in both PCR products and corresponds to the sequence of primers 2 and 3 ("internal primers"), which are complementary to each other and carry the "mismatch," i.e. the mutation. In the third PCR, the purified products of the previous two reactions are employed as template. Following denaturation, a small fraction of the template DNA will anneal to form heteroduplexes and will be extended at its recessed 3′ ends by the polymerase used in the reaction. The full-length sequence containing the mutation is then amplified using primers 1 and 4 ("external primers"). This mutant was produced using as external primers 5′-ATCGGCTCGTATAATGTGTGG-3′ and 5′-GTTCTGATTTAATCTGTATCAGG-3′. Internal primers were 5′-GACCCCGTGTTCTTAAAGCAC-3′ and 5′-GTGCTTTAAGAACACGGGGTC-3′. The newly inserted part of the expression construct, pKKDDC-mutant, was sequenced to confirm mutation, and the plasmid was used to transform Escherichia coli SVS370. The conditions used for expression and purification of the mutant protein in E. coli (SVS370) were as described for the wild-type enzyme (2Bertoldi M. Moore P.S. Maras B. Dominici P. Borri Voltattorni C. J. Biol. Chem. 1996; 271: 23954-23959Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 11Moore P.S. Dominici P. Borri Voltattorni C. Biochem. J. 1996; 315: 249-256Crossref PubMed Scopus (38) Google Scholar). Since the mutant enzyme does not show detectable decarboxylase activity in the standard spectrophotometric assay, which measures production of aromatic amines, screening with antibodies to native DDC was therefore necessary to monitor the purification procedure. The purified mutant was homogenous as indicated by a single band on SDS-PAGE. The enzyme concentration was determined by using an εM of 1.3 × 105m−1cm−1. PLP content of holoDDC enzymes was determined by releasing the coenzyme in 0.1 m NaOH and by using εM = 6600m−1 cm−1 at 388 nm. A sample of 20 μg of protein was subjected to SDS-polyacrylamide gel electrophoresis using a 12.5% acrylamide gel. The proteins were electroblotted to Immobilon-P-membranes (Millipore), and Western blot analysis was performed according to Gallagher et al. (12Gallagher S. Winston S.E. Fuller S.A. Hurrel J.G.R. Current Protocols in Molecular Biology. Greene Publishing and Wiley-Interscience, New York1993: 10.8.1-10.8.17Google Scholar). DDC mutant Y332F (2–5 μm) was incubated with (0.1–5 mm) l-Dopa orl-5-HTP in 50 mm Hepes, pH 7.5, at 25 °C in the presence or absence of O2. Production of dopamine or 5-HT was determined with a spectrophotometric assay outlined by Sheraldet al. (13Sherald A.F. Sparrow J.C. Wright T.R.F. Anal. Biochem. 1973; 56: 300-305Crossref PubMed Scopus (60) Google Scholar) and modified by Charteris and John (14Charteris A. John R.A. Anal. Biochem. 1975; 66: 365-371Crossref PubMed Scopus (63) Google Scholar). Alternatively, production of aromatic amines as well as consumption ofl-aromatic amino acids were measured by HPLC analysis. Aliquots were removed at time intervals, and trichloroacetic acid was added to a final concentration of 5% (v/v). The quenched solutions were centrifuged to remove protein, and the supernatants were analyzed using a Discovery (Supelco) C18 column (4.6 × 250 mm). The eluent was methanol:acetic acid:H2O, 24:1:75 with 6 mmoctanesulfonic acid at a flow rate of 0.6 ml/min. Detection was set at 280 nm. The concentration of l-aromatic amino acids and aromatic amines in the analyzed samples was determined from a standard curve generated from known concentrations of the compounds with respect to the internal standard. The amounts of ammonia and aromatic aldehyde (produced during the reaction of the Y332F mutant withl-aromatic amino acids or aromatic amines) were determined using the coupled assays with glutamate dehydrogenase and alcohol dehydrogenase, respectively, as described (2Bertoldi M. Moore P.S. Maras B. Dominici P. Borri Voltattorni C. J. Biol. Chem. 1996; 271: 23954-23959Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The amount of aldehyde or ammonia was measured by the decrease in absorbance at 340 nm due to the conversion of NADH to NAD+. The rate of production of14CO2 during the reaction of mutant with [1-14C]Dopa was determined as described (4Bertoldi M. Borri Voltattorni C. Biochem. J. 2000; 352: 533-538Crossref PubMed Scopus (38) Google Scholar). Radioactivity was determined with a Beckman Instruments LS 1801 liquid scintillation counter. H2O2 production and O2 consumption were measured according to Refs 1Bertoldi M. Frigeri P. Paci M. Borri Voltattorni C. J. Biol. Chem. 1999; 274: 5514-5521Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar and3Bertoldi M. Dominici P. Moore P.S. Maras B. Borri Voltattorni C. Biochemistry. 1998; 37: 6552-6561Crossref PubMed Scopus (40) Google Scholar, respectively. The detection and quantification of PLP and PMP content were performed using the HPLC procedure described previously (1Bertoldi M. Frigeri P. Paci M. Borri Voltattorni C. J. Biol. Chem. 1999; 274: 5514-5521Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 4Bertoldi M. Borri Voltattorni C. Biochem. J. 2000; 352: 533-538Crossref PubMed Scopus (38) Google Scholar). The apparent pseudo first-order rate constants,k obs, of the reaction of the Y332F mutant at varying concentrations of d-5-HTP were obtained by measuring the decrease of the 425-nm absorbance band as described (1Bertoldi M. Frigeri P. Paci M. Borri Voltattorni C. J. Biol. Chem. 1999; 274: 5514-5521Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The dependence of k obs on d-5HTP concentrations exhibits a saturation behavior, and a hyperbolic fit gives the value of apparent dissociation constant,K D, and the maximum value of rate constant, kmax. Enzymic assays were performed under anaerobic conditions with 1 ml of Reacti-Vials (Aldrich) as described previously (3Bertoldi M. Dominici P. Moore P.S. Maras B. Borri Voltattorni C. Biochemistry. 1998; 37: 6552-6561Crossref PubMed Scopus (40) Google Scholar, 4Bertoldi M. Borri Voltattorni C. Biochem. J. 2000; 352: 533-538Crossref PubMed Scopus (38) Google Scholar). Absorption spectra were recorded in a Jasco V-550 spectrophotometer. CD measurements were carried out in a Jasco J-710 spectropolarimeter at a scan speed of 50 nm/min with a bandwidth of 2 nm. Like the wild type, the mutant binds 2 mol of PLP per dimer. Absorption and CD spectra of the Y332F mutant in the UV-visible and far UV region are essentially identical to those of the wild-type enzyme (data not shown). When the Y332F mutant was incubated at 25 °C under aerobic conditions withl-Dopa, no dopamine formation was detected either by the spectrophotometric or the HPLC assays. However, l-Dopa level decreases, and its decrease parallels the production of 3,4-dihydroxyphenylacetaldehyde and ammonia accompanied by O2 consumption in a 1:2 molar ratio with respect to the products (Fig. 2). When the reaction was performed under the same experimental conditions in the presence of [1-14C]Dopa, 14CO2 release was observed. The initial velocities of these catalytic events are reported in Table I. Likewise, although no 5-HT production could be detected during the reaction of Y332F withl-5-HTP, conversion of l-5HTP into 5-hydroxyindolacetaldehyde and ammonia as well as consumption of O2 in a 1:2 molar ratio with respect to the products take place with the initial velocity values reported in Table I. During the reaction of Y332F, either with l-Dopa or withl-5-HTP, no detectable H2O2 was found. Initial velocities of oxidase activity, measured asl-aromatic amino acid consumption at varying concentrations of l-Dopa or l-5-HTP, have been determined. Thek cat and K m values are reported in Table II. As for the wild type, upon addition of l-Dopa to Y332F, an increased absorption centered at 425 nm immediately appears. This absorbance band, attributed to the external aldimine, decreases with time, and after the time required for consumption of substrate, the original 425-nm absorbance of the holoenzyme reappears (Fig.3). Qualitatively identical spectral changes are observed upon addition of l-5-HTP to the mutant. When the reaction of Y332F with l-Dopa orl-5-HTP was performed under anaerobic conditions, the concentration of the substrates remained almost unchanged with time, and neither dopamine nor 5-HT were produced. Instead, a conversion of PLP into PMP takes place very quickly: at 30 s, 89% (forl-Dopa) and 55% (for l-5-HTP) of the original coenzyme content are transformed into PMP.Table IInitial velocities of reaction of Y332F mutant (2.4 μm) with 200 μm l-Dopa or l-5-HTPSubstrate or productl-Dopal-5-HTPAromatic amineNot detectedNot detectedl-Aromatic amino acid0.83 ± 0.040.063 ± 0.004Aromatic aldehyde0.72 ± 0.030.053 ± 0.003Ammonia0.75 ± 0.040.055 ± 0.002O20.39 ± 0.010.031 ± 0.00214CO20.82 ± 0.03Not determinedNumbers represent initial velocities expressed as mol/sec/mol enzyme. Data shown are means of three independent experiments; S.E. mean in each case was less than 6% of the mean value. Open table in a new tab Table IIApparent kinetic properties for wild-type DDC and Y332F mutantReactionWild typeY332Fk cat ork maxK m orK Dk cat/K mork max/K Dk catork maxK Dk cat/K mor k max/K Dmin −1mmmin−1mm−1min −1mmmin−1mm−1Oxidative deaminationl-DopaNDNDND280 ± 460.72 ± 0.03389 ± 66l-5-HTPNDNDND63 ± 16.8 ± 0.59.3 ± 0.75-HT1.26 ± 0.042-aValues are from Ref. 5.0.6 ± 0.12-aValues are from Ref. 5.2.1 ± 0.32-aValues are from Ref. 5.0.71 ± 0.065.0 ± 0.90.14 ± 0.03Dopamine2.49 ± 0.071.8 ± 0.11.38 ± 0.081.7 ± 0.222 ± 50.08 ± 0.02Half-transaminationD-5-HTP0.086 ± 0.0032-bValues are from Ref. 1.1.1 ± 0.22-bValues are from Ref. 1.0.08 ± 0.012-bValues are from Ref. 1.0.058 ± 0.00112.5 ± 0.70.0046 ± 0.0003Decarboxylationl-Dopa256 ± 132-cValues are from Ref. 4.0.070 ± 0.0052-cValues are from Ref. 4.3657 ± 3202-cValues are from Ref. 4.NDNDNDl-5-HTP119 ± 192-cValues are from Ref. 4.0.15 ± 0.012-cValues are from Ref. 4.793 ± 1372-cValues are from Ref. 4.NDNDNDOxidative deaminase activity was determined by measuring either production of aromatic aldehyde or ammonia or consumption ofl-aromatic amino acids (see Experimental Procedures). Half-transaminase activity was determined by measuring the rate constants of the 425-nm absorbance decrease at varying D-5-HTP concentrations, as described under "Experimental Procedures." Decarboxylase activity was determined by measuring production of aromatic amines, as described under "Experimental Procedures."ND, not detected. k cat and K mwere obtained from nonlinear regression fit to the Michaelis-Menten equation using SigmaPlot2000 (SPSS). The errors reported are the standard error values derived by the curve fitting program.2-a Values are from Ref. 5Bertoldi M. Borri Voltattorni C. Protein Sci. 2001; 10: 1178-1186Crossref PubMed Scopus (15) Google Scholar.2-b Values are from Ref. 1Bertoldi M. Frigeri P. Paci M. Borri Voltattorni C. J. Biol. Chem. 1999; 274: 5514-5521Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar.2-c Values are from Ref. 4Bertoldi M. Borri Voltattorni C. Biochem. J. 2000; 352: 533-538Crossref PubMed Scopus (38) Google Scholar. Open table in a new tab Figure 3Time-dependent absorbance changes occurring upon addition ofl-Dopa to Y332F mutant. The enzyme (2.9 μm) (dashed line) in 50 mm Hepes, pH 7.5, was treated with l-Dopa (200 μm), and spectra were recorded at the indicated times.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Numbers represent initial velocities expressed as mol/sec/mol enzyme. Data shown are means of three independent experiments; S.E. mean in each case was less than 6% of the mean value. Oxidative deaminase activity was determined by measuring either production of aromatic aldehyde or ammonia or consumption ofl-aromatic amino acids (see Experimental Procedures). Half-transaminase activity was determined by measuring the rate constants of the 425-nm absorbance decrease at varying D-5-HTP concentrations, as described under "Experimental Procedures." Decarboxylase activity was determined by measuring production of aromatic amines, as described under "Experimental Procedures." ND, not detected. k cat and K mwere obtained from nonlinear regression fit to the Michaelis-Menten equation using SigmaPlot2000 (SPSS). The errors reported are the standard error values derived by the curve fitting program. Despite its total impairment of aromatic amines generation, the Y332F mutant retains substantial catalytic competence for the oxidative deamination of aromatic amines. In fact, reaction of the Y332F mutant with 5-HT or dopamine produces the corresponding aromatic aldehyde (5-hydroxyindolacetaldehyde or 3, 4-dihydroxyphenylacetaldehyde) and ammonia in equivalent amounts and consumes O2. On the other hand, aromatic amines undergo half-transamination under anaerobic conditions with rate constants similar to those of wild type (< 0.1 min−1) (1Bertoldi M. Frigeri P. Paci M. Borri Voltattorni C. J. Biol. Chem. 1999; 274: 5514-5521Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 5Bertoldi M. Borri Voltattorni C. Protein Sci. 2001; 10: 1178-1186Crossref PubMed Scopus (15) Google Scholar). Reaction of d-5-HTP with the mutant results in time-dependent spectral changes consisting of a decrease of the 425-nm absorbance band and a concomitant increase in the 330-nm region. These spectral changes correspond to conversion of PLP-bound to PMP (data not shown). This behavior is identical to that already observed with wild type (1Bertoldi M. Frigeri P. Paci M. Borri Voltattorni C. J. Biol. Chem. 1999; 274: 5514-5521Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 2Bertoldi M. Moore P.S. Maras B. Dominici P. Borri Voltattorni C. J. Biol. Chem. 1996; 271: 23954-23959Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 3Bertoldi M. Dominici P. Moore P.S. Maras B. Borri Voltattorni C. Biochemistry. 1998; 37: 6552-6561Crossref PubMed Scopus (40) Google Scholar). Steady-state kinetic parameters were determined for both oxidative deaminase and half-transaminase activities and reported in Table II. To allow a comparison, the values of the wild-type DDC are also included. The Y332F mutant shows negligible changes from the wild type in thek cat value for oxidative deamination and in the k max of half-transamination but has increased K m values (for aromatic amines) and increased K D values (for d-5-HTP) as compared with those of the wild-type enzyme. Flexible loops that occlude active sites during catalysis are recognized as structural elements common to many enzymes. Loop closure induced by substrate binding has great importance in catalysis and specificity by recruiting functional groups into the active site (15Clarke A. Wigley D.B. Chia W.N. Barstow D. Atkinson T. Holbrook J.J. Nature. 1986; 324: 699-702Crossref PubMed Scopus (161) Google Scholar, 16Krell T. MacLean J. Boam D.J. Cooper A. Resmini M. Brocklehurst K. Kelly S.M. Price N.C. Lapthorn A.J. Coggins J.R. Protein Sci. 2001; 10: 1137-1149Crossref PubMed Scopus (67) Google Scholar, 17Zgiby S. Plater A.R. Bates M.A. Thomson G.J. Berry A. J. Mol. Biol. 2002; 315: 131-140Crossref PubMed Scopus (42) Google Scholar), stabilizing reactive intermediates (18Pompliano D.L. Peyman A. Knowles J.R. Biochemistry. 1990; 29: 3186-3194Crossref PubMed Scopus (254) Google Scholar, 19Larson E.M. Larimer F.W. Hartman F.C. Biochemistry. 1995; 34: 4531-4537Crossref PubMed Scopus (32) Google Scholar, 20Kato H. Tanaka T. Yamaguchi H. Hara T. Nishioka T. Katsube Y. Oda J. Biochemistry. 1994; 33: 4995-4999Crossref PubMed Scopus (44) Google Scholar), and preventing the formation of stable abortive complexes (21Contestabile R. Angelaccio S. Maytum R. Bossa F. John R.A. J. Biol. Chem. 2000; 275: 3879-3886Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Kinetic and structural data have indicated that an 11-residue loop in DDC is conformationally dynamic, suggesting that this loop closes over the active site after substrate binding. This study was prompted to gain insights into the function of the mobile residue of this loop, Tyr-332. Replacement of Tyr-332 with phenylalanine results in a protein that is still capable of selectively cleaving the correct bond between Cα and COOH of l-aromatic amino acids. This is supported by the finding that during the reaction of the mutant with l-Dopa in aerobiosis, CO2 is released, and by the occurrence in anaerobiosis of a decarboxylation-dependent transamination of both l-Dopa and l-5-HTP. In fact, PMP formation would be promoted along the reaction pathway if the decarboxylated substrate (quinoid) intermediate is protonated at C4′ instead at Cα. On the basis of these data, it can be anticipated that DDC does not require Tyr-332 for the decarboxylation step. Nevertheless, the Y332F mutant is unable to generate aromatic amines to any discernible extent. Since reprotonation at Cα after decarboxylation is necessary for the generation of aromatic amines (Scheme FS1 (a)), a role for Tyr-332 residue as a proton donor in this reprotonation step can be advanced. However, no accumulation of quinonoid intermediate during the reaction of the Y332F mutant with l-aromatic amino acids can be observed. This would be explained by the fact that, although the original overall decarboxylation of the wild-type enzyme is completely suppressed, a new catalytic activity is generated in the presence of O2. It consists of a decarboxylation-dependent oxidative deamination converting l-aromatic amino acids into CO2, aromatic aldehydes, and ammonia occurring with a consumption of molecular oxygen in a 1:2 molar ratio with respect to the products. An oxidase activity displaying the same stoichiometry has been already reported to be catalyzed by the wild-type enzyme toward aromatic amines (2Bertoldi M. Moore P.S. Maras B. Dominici P. Borri Voltattorni C. J. Biol. Chem. 1996; 271: 23954-23959Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 3Bertoldi M. Dominici P. Moore P.S. Maras B. Borri Voltattorni C. Biochemistry. 1998; 37: 6552-6561Crossref PubMed Scopus (40) Google Scholar). Analogous with the already proposed mechanism for the latter reaction (5Bertoldi M. Borri Voltattorni C. Protein Sci. 2001; 10: 1178-1186Crossref PubMed Scopus (15) Google Scholar), it is reasonable to postulate that decarboxylation-dependent oxidative deamination ofl-aromatic amino acids catalyzed by the Y332F mutant occurs according to the mechanism outlined in Scheme FS1 (b). After release of CO2, binding of O2 to the C4′ of the quinonoid intermediate could lead to the formation of a peroxide anion that, once stabilized by protonation to the hydroperoxyPLP intermediate, will undergo heterolysis of the O–O bond. This allows regeneration of PLP and formation of an imine complex that spontaneously decomposes to aromatic aldehyde and ammonia. According to this mechanism, reaction of Y332F with l-aromatic amino acids does not occur through a serial mechanism in which aromatic amine, produced by decarboxylation, is a transient intermediate that is subsequently converted into aldehyde and ammonia. This is consistent with our data. In fact, the k cat values for oxidative deamination of aromatic amines catalyzed by the Y332F mutant, similar to those catalyzed by the wild-type DDC, are about 150-fold lower than those catalyzed by the mutant toward the correspondingl-aromatic amino acids (Table II). Moreover, reaction of the Y332F mutant with l-aromatic amino acids under anaerobic conditions is characterized by no accumulation of aromatic amine. In an O2-free atmosphere, if the amine were produced, it would accumulate since it would undergo a very slow transamination. Under anaerobic conditions, the C4′of the quinonoid intermediate cannot be oxygenated, but it could be protonated, giving rise to a ketimine substrate intermediate that would yield by hydrolysis the PMP enzyme and the carbonyl compound (Scheme FS1 (c)). This is consistent with our data in the absence of O2. It should be noted that whereas in anaerobiosis for the Y332F mutant, the abortive transamination represents the 100% of the catalytic events, for wild-type DDC, it takes place at a ratio of about once per 5 × 103 and 6.5 × 103 times of decarboxylation for l-Dopa and l-5-HTP, respectively (4Bertoldi M. Borri Voltattorni C. Biochem. J. 2000; 352: 533-538Crossref PubMed Scopus (38) Google Scholar). All together, these results indicate that since the quinonoid intermediate in Y332F cannot undergo protonation at Cα, its only possible fate is oxygenation or protonation at C4′ in the presence or absence, respectively, of O2. Since Tyr-332 likely plays a role as proton donor to Cα along the reaction pathway of decarboxylation, it is not surprising that thek cat value for the oxidative deamination and half-transamination reactions catalyzed by the Y332F mutant toward aromatic amines and d-5-HTP, respectively, remains largely unchanged with respect to the corresponding reactions catalyzed by the wild type. However, the mutant exhibits an ∼10-fold increase in theK m values for amines and in theK D values for d-5-HTP, as compared with those of wild type. The kinetic parameters for the decarboxylation-dependent oxidative deamination indicate that, although replacement of Tyr-332 with phenylalanine led to 10-fold increase in K m value forl-Dopa, the k cat value is similar to that for the overall decarboxylation catalyzed by the wild type for the same substrate. More substantial changes were found whenl-5-HTP was used as substrate (40-fold increase inK m value and ∼2-fold decrease ink cat value for l-5HTP as compared with the wild type) (Table II). Thus, although the catalytic efficiencies of the reactions catalyzed by the Y332F mutant are lower than the corresponding ones for wild type, the major difference is reflected in K m values. This may be due to the fact that Y → F replacement at position 332 weakens the binding of substrates to the enzyme, although not precluding it. The substitution of phenylalanine for tyrosine 332 has changed the catalytic properties of DDC toward l-aromatic amino acids: the original overall decarboxylation is completely abolished, and a new catalytic activity not inherent in the wild type is generated. Few site-directed mutagenesis experiments altering the reaction specificity of PLP enzymes have been reported, even if a complete suppression of the original activity has never been achieved (22Vacca R. Christen P. Malashkevich V.N. Jansonius J.N. Sandmeier E. Eur. J. Biochem. 1995; 227: 481-487Crossref PubMed Scopus (17) Google Scholar, 23Graber R. Kasper P. Malashkevich V.N. Strop P. Gehring H. Jansonius J.J. Christen P. J. Biol. Chem. 1999; 274: 31203-31208Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 24Jackson J.N. Brooks H.B. Osterman A.L. Goldsmith E.J. Phillips M.A. Biochemistry. 2000; 39: 11247-11257Crossref PubMed Scopus (78) Google Scholar, 25Bertoldi M. Castellani S. Borri Voltattorni C. Eur. J. Biochem. 2001; 268: 2975-2981Crossref PubMed Scopus (23) Google Scholar). It is of interest that replacement of Cys-360 by Ala or Ser in eukaryotic ornithine decarboxylase greatly reduces the rate of decarboxylation and increases the rate of the abortive transamination. On this basis, a role for Cys-360 in facilitating decarboxylation has been proposed (24Jackson J.N. Brooks H.B. Osterman A.L. Goldsmith E.J. Phillips M.A. Biochemistry. 2000; 39: 11247-11257Crossref PubMed Scopus (78) Google Scholar). Likewise, mutation of residues in the coenzyme binding pocket of DDC alters the nature of catalysis by enhancing decarboxylation-dependent transamination activity and reducing original decarboxylation activity (25Bertoldi M. Castellani S. Borri Voltattorni C. Eur. J. Biochem. 2001; 268: 2975-2981Crossref PubMed Scopus (23) Google Scholar). To the best of our knowledge, such a clear change in reaction specificity of Y332F DDC with a remarkably high new activity (k cat values of 4.6 s−1 and 1.0 s−1 for l-Dopa and l-5HTP, respectively) has as of yet only been reported for papain that was converted into a peptide-nitrite hydratase by a single amino acid substitution at the active site (26Dufour E. Storer A.C. Mènard R. Biochemistry. 1995; 34: 16382-16388Crossref PubMed Scopus (53) Google Scholar). Strong evidence is provided that loop movement is critical for a correct positioning of Tyr-332 to allow its catalytic role in reprotonation of the quinonoid at Cα. This is the first PLP-α decarboxylase for which the residue responsible for the protonation of the decarboxylated reaction intermediate to form physiological amines has been identified.