Three different classes of aminotransferases evolved prephenate aminotransferase functionality in arogenate-competent microorganisms.
ABSTRACT: The aromatic amino acids phenylalanine and tyrosine represent essential sources of high value natural aromatic compounds for human health and industry. Depending on the organism, alternative routes exist for their synthesis. Phenylalanine and tyrosine are synthesized either via phenylpyruvate/4-hydroxyphenylpyruvate or via arogenate. In arogenate-competent microorganisms, an aminotransferase is required for the transamination of prephenate into arogenate, but the identity of the genes is still unknown. We present here the first identification of prephenate aminotransferases (PATs) in seven arogenate-competent microorganisms and the discovery that PAT activity is provided by three different classes of aminotransferase, which belong to two different fold types of pyridoxal phosphate enzymes: an aspartate aminotransferase subgroup 1? in tested ?- and ?-proteobacteria, a branched-chain aminotransferase in tested cyanobacteria, and an N-succinyldiaminopimelate aminotransferase in tested actinobacteria and in the ?-proteobacterium Nitrosomonas europaea. Recombinant PAT enzymes exhibit high activity toward prephenate, indicating that the corresponding genes encode bona fide PAT. PAT functionality was acquired without other modification of substrate specificity and is not a general catalytic property of the three classes of aminotransferases.
Project description: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.
Project description:In addition to being a vital component of proteins, phenylalanine is also a precursor of numerous aromatic primary and secondary metabolites with broad physiological functions. In plants phenylalanine is synthesized predominantly via the arogenate pathway in plastids. Here, we describe the structure, molecular players and subcellular localization of a microbial-like phenylpyruvate pathway for phenylalanine biosynthesis in plants. Using a reverse genetic approach and metabolic flux analysis, we provide evidence that the cytosolic chorismate mutase is responsible for directing carbon flux towards cytosolic phenylalanine production via the phenylpyruvate pathway. We also show that an alternative transcription start site of a known plastidial enzyme produces a functional cytosolic prephenate dehydratase that catalyzes the conversion of prephenate to phenylpyruvate, the intermediate step between chorismate mutase and phenylpyruvate aminotransferase. Thus, our results complete elucidation of phenylalanine biosynthesis via phenylpyruvate in plants, showing that this pathway splits from the known plastidial arogenate pathway at chorismate, instead of prephenate as previously thought, and the complete pathway is localized in the cytosol.
Project description:TyrA proteins belong to a family of dehydrogenases that are dedicated to l-tyrosine biosynthesis. The three TyrA subclasses are distinguished by their substrate specificities, namely the prephenate dehydrogenases, the arogenate dehydrogenases, and the cyclohexadienyl dehydrogenases, which utilize prephenate, l-arogenate, or both substrates, respectively. The molecular mechanism responsible for TyrA substrate selectivity and regulation is unknown. To further our understanding of TyrA-catalyzed reactions, we have determined the crystal structures of Aquifex aeolicus prephenate dehydrogenase bound with NAD(+) plus either 4-hydroxyphenylpyuvate, 4-hydroxyphenylpropionate, or l-tyrosine and have used these structures as guides to target active site residues for site-directed mutagenesis. From a combination of mutational and structural analyses, we have demonstrated that His-147 and Arg-250 are key catalytic and binding groups, respectively, and Ser-126 participates in both catalysis and substrate binding through the ligand 4-hydroxyl group. The crystal structure revealed that tyrosine, a known inhibitor, binds directly to the active site of the enzyme and not to an allosteric site. The most interesting finding though, is that mutating His-217 relieved the inhibitory effect of tyrosine on A. aeolicus prephenate dehydrogenase. The identification of a tyrosine-insensitive mutant provides a novel avenue for designing an unregulated enzyme for application in metabolic engineering.
Project description:In plants, the amino acids tyrosine and phenylalanine are synthesized from arogenate by arogenate dehydrogenase and arogenate dehydratase, respectively, with the relative flux to each being tightly controlled. Here the characterization of a maize opaque endosperm mutant (mto140), which also shows retarded vegetative growth, is described The opaque phenotype co-segregates with a Mutator transposon insertion in an arogenate dehydrogenase gene (zmAroDH-1) and this led to the characterization of the four-member family of maize arogenate dehydrogenase genes (zmAroDH-1-zmAroDH-4) which share highly similar sequences. A Mutator insertion at an equivalent position in AroDH-3, the most closely related family member to AroDH-1, is also associated with opaque endosperm and stunted vegetative growth phenotypes. Overlapping but differential expression patterns as well as subtle mutant effects on the accumulation of tyrosine and phenylalanine in endosperm, embryo, and leaf tissues suggest that the functional redundancy of this gene family provides metabolic plasticity for the synthesis of these important amino acids. mto140/arodh-1 seeds shows a general reduction in zein storage protein accumulation and an elevated lysine phenotype typical of other opaque endosperm mutants, but it is distinct because it does not result from quantitative or qualitative defects in the accumulation of specific zeins but rather from a disruption in amino acid biosynthesis.
Project description:Prephenate is the direct precursor of phenylpyruvate and 4-hydroxyphenylpyruvate in the biogenesis of phenylalanine and tyrosine by action of the decarboxylative, aromatizing enzymes prephenate dehydratase and dehydrogenase, respectively. The recent characterization of BacA in bacilysin biosynthesis as a nonaromatizing decarboxylase reveals a new route from prephenate in the biosynthesis of nonproteinogenic amino acids. This study describes two additional enzymes, AerD from Planktothrix agardhii and SalX from Salinispora tropica, that utilize the central building block prephenate for flux down distinct pathways to amino acid products, representing a new metabolic fate for prephenate and establishing a new family of nonaromatizing prephenate decarboxylases.
Project description:Arogenate dehydratases (ADTs) catalyze the final step in phenylalanine biosynthesis in plants. The Arabidopsis thaliana genome encodes a family of six ADTs capable of decarboxylating/dehydrating arogenate into phenylalanine. Using cyan fluorescent protein (CFP)-tagged proteins, the subcellular localization patterns of all six A. thaliana ADTs were investigated in intact Nicotiana benthamiana and A. thaliana leaf cells. We show that A. thaliana ADTs localize to stroma and stromules (stroma-filled tubules) of chloroplasts. This localization pattern is consistent with the enzymatic function of ADTs as many enzymes required for amino acid biosynthesis are primarily localized to chloroplasts, and stromules are thought to increase metabolite transport from chloroplasts to other cellular compartments. Furthermore, we provide evidence that ADTs have additional, non-enzymatic roles. ADT2 localizes in a ring around the equatorial plane of chloroplasts or to a chloroplast pole, which suggests that ADT2 is a component of the chloroplast division machinery. In addition to chloroplasts, ADT5 was also found in nuclei, again suggesting a non-enzymatic role for ADT5. We also show evidence that ADT5 is transported to the nucleus via stromules. We propose that ADT2 and ADT5 are moonlighting proteins that play an enzymatic role in phenylalanine biosynthesis and a second role in chloroplast division or transcriptional regulation, respectively.
Project description:Prephenate dehydratase (PDT) is a key regulatory enzyme in L-phenylalanine biosynthesis in the Gram-positive bacterium Amycolatopsis methanolica. The PDT protein was purified to homogeneity (1957-fold) from wild-type cells with a final yield of 6.5%. It was characterized as a 150 kDa homotetrameric protein with a subunit size of 34 kDa. The first 35 N-terminal amino acids were identified, revealing highest similarity to the PDT proteins from Corynebacterium glutamicum and Bacillus subtilis. Kinetic studies showed that the A. methanolica PDT is allosterically inhibited by phenylalanine and activated by tyrosine. Phenylalanine caused an increase in the S0.5 for prephenate and a decrease in the Vmax. Tyrosine caused a decrease in the S0.5 for prephenate and an increase in the Vmax. Spontaneous o-fluoro- and p-fluoro-DL-phenylalanine-resistant mutants of A. methanolica were isolated. Kinetic studies with the partially purified PDT proteins of strains pFPhe32 and oFPhe84 showed that these mutant proteins had become (partly) insensitive to both phenylalanine inhibition and tyrosine activation.
Project description:Chorismate mutase/prephenate dehydrogenase from Haemophilus influenzae Rd KW20 is a bifunctional enzyme that catalyzes the rearrangement of chorismate to prephenate and the NAD(P)(+)-dependent oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate in tyrosine biosynthesis. The crystal structure of the prephenate dehydrogenase component (HinfPDH) of the TyrA protein from H. influenzae Rd KW20 in complex with the inhibitor tyrosine and cofactor NAD(+) has been determined to 2.0?Å resolution. HinfPDH is a dimeric enzyme, with each monomer consisting of an N-terminal ?/? dinucleotide-binding domain and a C-terminal ?-helical dimerization domain. The structure reveals key active-site residues at the domain interface, including His200, Arg297 and Ser179 that are involved in catalysis and/or ligand binding and are highly conserved in TyrA proteins from all three kingdoms of life. Tyrosine is bound directly at the catalytic site, suggesting that it is a competitive inhibitor of HinfPDH. Comparisons with its structural homologues reveal important differences around the active site, including the absence of an ?-? motif in HinfPDH that is present in other TyrA proteins, such as Synechocystis sp. arogenate dehydrogenase. Residues from this motif are involved in discrimination between NADP(+) and NAD(+). The loop between ?5 and ?6 in the N-terminal domain is much shorter in HinfPDH and an extra helix is present at the C-terminus. Furthermore, HinfPDH adopts a more closed conformation compared with TyrA proteins that do not have tyrosine bound. This conformational change brings the substrate, cofactor and active-site residues into close proximity for catalysis. An ionic network consisting of Arg297 (a key residue for tyrosine binding), a water molecule, Asp206 (from the loop between ?5 and ?6) and Arg365' (from the additional C-terminal helix of the adjacent monomer) is observed that might be involved in gating the active site.
Project description:Buchnera aphidicola, the prokaryotic endosymbiont of aphids, complements dietary deficiencies with the synthesis and provision of several essential amino acids. We have cloned and sequenced a region of the genome of B. aphidicola isolated from Acyrthosiphon pisum which includes the two-domain aroQ/pheA gene. This gene encodes the bifunctional chorismate mutase-prephenate dehydratase protein, which plays a central role in L-phenylalanine biosynthesis. Two changes involved in the overproduction of this amino acid have been detected. First, the absence of an attenuator region suggests a constitutive expression of this gene. Second, the regulatory domain of the Buchnera prephenate dehydratase shows changes in the ESRP sequence, which is involved in the allosteric binding of phenylalanine and is strongly conserved in prephenate dehydratase proteins from practically all known organisms. These changes suggest the desensitization of the enzyme to inhibition by phenylalanine and would permit the bacterial endosymbiont to overproduce phenylalanine.
Project description:L-Tyrosine (Tyr) is an aromatic amino acid synthesized de novo in plants and microbes. In animals, Tyr must be obtained through their diet or synthesized from L-phenylalanine. In addition to protein synthesis, Tyr serves as the precursor of neurotransmitters (e.g., dopamine and epinephrine) in animals and of numerous plant natural products, which serve essential functions in both plants and humans (e.g., vitamin E and morphine). Tyr is synthesized via two alternative routes mediated by a TyrA family enzyme, prephenate, or arogenate dehydrogenase (PDH/TyrAp or ADH/TyrAa), typically found in microbes and plants, respectively. Although ADH activity is also found in some bacteria, the origin of arogenate-specific TyrAa enzymes is unknown. We recently identified an acidic Asp222 residue that confers ADH activity in plant TyrAs. In this study, structure-guided phylogenetic analyses identified bacterial homologs, closely-related to plant TyrAs, that also have an acidic 222 residue and ADH activity. A more distant archaeon TyrA that preferred PDH activity had a non-acidic Gln, whose substitution to Glu introduced ADH activity. These results indicate that the conserved molecular mechanism operated during the evolution of arogenate-specific TyrAa in both plants and microbes.