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Tyrosine depletion in metazoan proteins was recently explained to be due to the appearance of tyrosine kinases in Metazoa. Here, we present a complementary explanation for the depletion of tyrosine, stating the importance of tyrosine in signaling not only as a phosphorylation target, but also as a precursor for catecholamines and hormones. Molecules (dopamine, norepinephrine, and epinephrine, and to a lesser extent serotonin and melatonin) critical to metazoan multicellular signaling are also greatly dependent on a supply of tyrosine. These signaling molecules are synthesized in two highly linked pathways specific to metazoans. In addition, the shikimate pathway that non-metazoans use to synthesize the aromatic amino acids is not present in metazoans. These important pathway changes have occurred between Metazoa and other eukaryotes, causing significant changes to tyrosine metabolism and rendering tyrosine crucial for extracellular signaling. Additionally, the evolutionary and functional linkage between these two pathways and the resulting implications for neuropathology are discussed.

Phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of L-Phe to L-Tyr. Dysfunctional PAH results in phenylketonuria and mammalian PAH is therefore highly regulated and displays positive cooperativity for L-Phe (Hill coefficient (h)=2). L-Phe does not bind to the regulatory ACT domain in full-length tetrameric human PAH and cooperativity is elicited by homotropic binding to the catalytic site (Thórólfsson et al. in Biochemistry 41:7573-7585, 2002). PAH from Caenorhabditis elegans (cePAH) is devoid of cooperativity for L-Phe (h=0.9), and, as shown in this work, structural analysis reveal an additional L-Phe binding site at the regulatory domain of full-length cePAH. This site involves the GA(S)L/ISRP motifs, which are also found in ACT domains of other L-Phe binding proteins, such as prephenate dehydratase. Isothermal titration calorimetry further demonstrated 2 binding sites per subunit for cePAH versus ~1 for hPAH. Steric occlusion of the regulatory site, notably by residues Lys215/Tyr216 from the adjacent catalytic domain, appears to hinder regulatory binding in full-length hPAH. Accordingly, the humanized mutant Q215K/N216Y of cePAH binds ~1.4 L-Phe/subunit. This mutant also displays high catalytic activity and certain positive cooperativity for L-Phe (h=1.4). Our results support that the acquisition of positive cooperativity in mammalian forms of PAH is accompanied by a closure of the regulatory L: -Phe binding site. Concomitantly, the function of the regulatory ACT domain appears to be adapted from amino acid binding to serving the communication of conformational changes among catalytic subunits.

High sequence divergence, evolutionary mobility, and superfold topology characterize the ACT domain. Frequently found in multidomain proteins, these domains induce allosteric effects by binding a regulatory ligand usually to an ACT domain dimer interface. In mammalian phenylalanine hydroxylase (PAH), no contacts are formed between ACT domains, and the domain promotes an allosteric effect despite the apparent lack of ligand binding. The increased functional scenario of this abundant domain encouraged us to search for distant homologs, aiming to enhance the understanding of the ACT domain in general and the ACT domain of PAH in particular. The PDB was searched using the FATCAT server with the ACT domain of PAH as a query. The hits that were confirmed by the SSAP algorithm were divided into known ACT domains (KADs) and potential ACT domains (PADs). The FATCAT/SSAP procedure recognized most of the established KADs, as well 18 so far unrecognized non-redundant PADs with extremely low sequence identities and high divergence in functionality and oligomerization. However, analysis of the structural similarity provides remarkable clustering of the proteins according to similarities in ligand binding. Despite enormous sequence divergence and high functional variability, there is a common regulatory theme among these domains. The results reveal the close relationships of the ACT domain of PAH with amino acid binding and metallobinding ACT domains and with acylphosphatase.