Publications

• Biochemical characterization and crystal structure of Synechocystis arogenate dehydrogenase provide insights into catalytic reaction.

  Legrand P., Dumas R., Seux M., Rippert P., Ravelli R., Ferrer J.L., Matringe M.

  The extreme diversity in substrate specificity, and in the regulation mechanism of arogenate/prephenate dehydrogenase enzymes in nature, makes a comparative structural study of these enzymes of great interest. We report here on the biochemical and structural characterization of arogenate dehydroge ... >> More

  The extreme diversity in substrate specificity, and in the regulation mechanism of arogenate/prephenate dehydrogenase enzymes in nature, makes a comparative structural study of these enzymes of great interest. We report here on the biochemical and structural characterization of arogenate dehydrogenase from Synechocystis sp. (TyrAsy). This work paves the way for the understanding of the structural determinants leading to diversity in substrate specificity, and of the regulation mechanisms of arogenate/prephenate dehydrogenases. The overall structure of TyrAsy in complex with NADP was refined to 1.6 A. The asymmetric unit contains two TyrAsy homodimers, with each monomer consisting of a nucleotide binding N-terminal domain and a particularly unique alpha-helical C-terminal dimerization domain. The substrate arogenate was modeled into the active site. The model of the ternary complex enzyme-NADP-arogenate nicely reveals at the atomic level the concerted mechanism of the arogenate/prephenate dehydrogenase reaction. << Less

  Structure 14:767-776(2006) [PubMed] [EuropePMC]

• Molecular and biochemical characterization of an Arabidopsis thaliana arogenate dehydrogenase with two highly similar and active protein domains.

  Rippert P., Matringe M.

  The present study reports the first molecular characterization of a plant arogenate dehydrogenase, the enzyme that catalyses the transformation of arogenate into tyrosine. The structure of the Arabidopsis thaliana tyrA gene is very peculiar since it encodes two highly similar, and putatively activ ... >> More

  The present study reports the first molecular characterization of a plant arogenate dehydrogenase, the enzyme that catalyses the transformation of arogenate into tyrosine. The structure of the Arabidopsis thaliana tyrA gene is very peculiar since it encodes two highly similar, and putatively active, protein domains. PCR analyses confirmed the existence of a transcript encoding the two protein domains. The complete coding sequence and sequences corresponding to the two separate domains were independently cloned into Escherichia coli mutant AT 2471 lacking prephenate dehydrogenase activity. Our results revealed that the three recombinant enzymes are active. They all exhibit a high specificity toward arogenate and NADP, and have very similar kinetic properties. << Less

  Plant Mol. Biol. 48:361-368(2002) [PubMed] [EuropePMC]

• A core catalytic domain of the TyrA protein family: arogenate dehydrogenase from Synechocystis.

  Bonner C.A., Jensen R.A., Gander J.E., Keyhani N.O.

  The TyrA protein family includes prephenate dehydrogenases, cyclohexadienyl dehydrogenases and TyrA(a)s (arogenate dehydrogenases). tyrA(a) from Synechocystis sp. PCC 6803, encoding a 30 kDa TyrA(a) protein, was cloned into an overexpression vector in Escherichia coli. TyrA(a) was then purified to ... >> More

  The TyrA protein family includes prephenate dehydrogenases, cyclohexadienyl dehydrogenases and TyrA(a)s (arogenate dehydrogenases). tyrA(a) from Synechocystis sp. PCC 6803, encoding a 30 kDa TyrA(a) protein, was cloned into an overexpression vector in Escherichia coli. TyrA(a) was then purified to apparent homogeneity and characterized. This protein is a model structure for a catalytic core domain in the TyrA superfamily, uncomplicated by allosteric or fused domains. Competitive inhibitors acting at the catalytic core of TyrA proteins are analogues of any accepted cyclohexadienyl substrate. The homodimeric enzyme was specific for L-arogenate (K(m)=331 microM) and NADP+ (K(m)=38 microM), being unable to substitute prephenate or NAD+ respectively. L-Tyrosine was a potent inhibitor of the enzyme (K(i)=70 microM). NADPH had no detectable ability to inhibit the reaction. Although the mechanism is probably steady-state random order, properties of 2',5'-ADP as an inhibitor suggest a high preference for L-arogenate binding first. Comparative enzymology established that both of the arogenate-pathway enzymes, prephenate aminotransferase and TyrA(a), were present in many diverse cyanobacteria and in a variety of eukaryotic red and green algae. << Less

  Biochem J 382:279-291(2004) [PubMed] [EuropePMC]

• Variable enzymological patterning in tyrosine biosynthesis as a means of determining natural relatedness among the Pseudomonadaceae.

  Byng G.S., Whitaker R.J., Gherna R.L., Jensen R.A.

  Enzymes of tyrosine biosynthesis (prephenate dehydrogenase and arogenate dehydrogenase) were characterized in 90 species currently classified within the genera Pseudomonas, Xanthomonas, and Alcaligenes. Variation in cofactor specificity and regulatory properties of the dehydrogenase proteins allow ... >> More

  Enzymes of tyrosine biosynthesis (prephenate dehydrogenase and arogenate dehydrogenase) were characterized in 90 species currently classified within the genera Pseudomonas, Xanthomonas, and Alcaligenes. Variation in cofactor specificity and regulatory properties of the dehydrogenase proteins allowed the separation of five groups. Taxa defined by enzymological patterning corresponded strikingly with the five ribosomal ribonucleic acid (rRNA) homology groups established via rRNA-deoxyribonucleic acid hybridization. rRNA homology groups I, IV, and V all lack activity for arogenate/nicotinamide adenine dinucleotide phosphate (NADP) dehydrogenase and separated on this criterion from groups II and III, which have the activity. Group II species possess arogenate dehydrogenase enzyme (reactive with either NAD or NADP) sensitive to feedback inhibition by tyrosine, thereby separating from group III species whose corresponding enzyme was totally insensitive to feedback inhibition. The presence of prephenate/NADP dehydrogenase in group IV defined its separation from groups I and V, which lack this enzyme activity. Group I species possess an arogenate/NAD dehydrogenase that was highly sensitive to inhibition by tyrosine and a prephenate/NAD dehydrogenase of relative insensitivity to tyrosine inhibition. The opposite pattern of sensitivity/insensitivity was seen in group V species. These dehydrogenase characterizations are highly reliable for the keying of a given species to one of the five rRNA homology groups. If necessary, other confirmatory assays can be included using other aromatic pathway enzymes. These results further document the validity and utility of the approach of comparative enzymology and allostery for classification of microorganisms. << Less

  J Bacteriol 144:247-257(1980) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification and kinetic analysis of the two recombinant arogenate dehydrogenase isoforms of Arabidopsis thaliana.

  Rippert P., Matringe M.

  The present study reports the first purification and kinetic characterization of two plant arogenate dehydrogenases (EC 1.3.1.43), an enzyme that catalyses the oxidative decarboxylation of arogenate into tyrosine in presence of NADP. The two Arabidopsis thaliana arogenate dehydrogenases TyrAAT1 an ... >> More

  The present study reports the first purification and kinetic characterization of two plant arogenate dehydrogenases (EC 1.3.1.43), an enzyme that catalyses the oxidative decarboxylation of arogenate into tyrosine in presence of NADP. The two Arabidopsis thaliana arogenate dehydrogenases TyrAAT1 and TyrAAT2 were overproduced in Escherichia coli and purified to homogeneity. Biochemical comparison of the two forms revealed that at low substrate concentration TyrAAT1 is four times more efficient in catalyzing the arogenate dehydrogenase reaction than TyrAAT2. Moreover, TyrAAT2 presents a weak prephenate dehydrogenase activity whereas TyrAAT1 does not. The mechanism of the dehydrogenase reaction catalyzed by these two forms has been investigated using steady-state kinetics. For both enzymes, steady-state velocity patterns are consistent with a rapid equilibrium, random mechanism in which two dead-end complexes, E-NADPH-arogenate and E-NADP-tyrosine, are formed. << Less

  Eur. J. Biochem. 269:4753-4761(2002) [PubMed] [EuropePMC]