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

Abstract

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 with L-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 Studies on the effect exerted by O 2 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 O 2 and is accompanied by a decarboxylation-dependent transamination (4), and Reaction 2 occurs at the same extent either in the presence or absence of O 2 (4). Reaction 3 does not occur in anaerobiosis and is replaced by half-transamination (1, 5). Tancini et al. (6) 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 (1). 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 (1). The finding that recombinant rat liver DDC lacking this loop is incompetent for decarboxylation supports this view (7).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 (8). 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 * This work was supported by funding from the Italian Ministero dell'Istruzione, dell'Università e Ricerca and from Consiglio Nazionale delle Ricerche (CNR) (to C. B. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This...

Plant Phenylacetaldehyde Synthase Is a Bifunctional Homotetrameric Enzyme That Catalyzes Phenylalanine Decarboxylation and Oxidation

“…Unlike in the reaction catalyzed by the native enzyme, the amine product cannot be detected in the reaction catalyzed by the mutant enzyme (46). Thus, mutant pig kidney DDC has catalytic properties very similar to those of PAAS.…”

“…However, this possibility also seems unlikely, as PAAS is closely related to DDCs (Fig. 3), and only a simple mutation is required to convert pig kidney DDC from a non-oxidative decarboxylase to a fully oxidative decarboxylase (46). The only cofactor present in the active site of pig kidney DDC is PLP.…”

“…Bertoldi et al (46) showed that mutation of Tyr 332 to Phe converts pig kidney DDC into a decarboxylation-dependent deaminase, in which the decarboxylation of amino acid substrate (L-Dopa) is stoichiometric with oxidation. Unlike in the reaction catalyzed by the native enzyme, the amine product cannot be detected in the reaction catalyzed by the mutant enzyme (46).…”

Structural Study Reveals That Ser-354 Determines Substrate Specificity on Human Histidine Decarboxylase

“…In the case of GAD, the mobility of the flexible loop of GAD65 promoted a side reaction, resulting in auto-inactivation (41). The position of an important tyrosine (corresponding to Tyr-334 in hHDC) has not been identified in the AroDC structure, and the substitution of the corresponding tyrosine with phenylalanine resulted in suppression of decarboxylation activities (42). Taken together, these findings indicate that the loop of PLP-dependent decarboxylases should be positioned near the substrate-binding site when this loop is involved in the catalytic reaction as a proton donor and lid for solvent shielding.…”

Open conformation of human DOPA decarboxylase reveals the mechanism of PLP addition to Group II decarboxylases

“…4. Notice that loop1, loop2 (in particular Phe-103), and the flexible loop all contain residues involved in substrate binding (25,30,31); indicating that the structural rearrangement described here and the enzyme catalytic activity are tightly interconnected. Furthermore, of the 23 point mutations of DDC identified in patients with various neurotransmitters disorders 16 are part of the structural determinants involved in the conformational change that we propose, or make direct interaction with them (Table S1).…”