Structure–Function Relationships in the Aromatic Amino Acid Hydroxylases Enzyme Family: Evolutionary Insights


The nonheme iron and tetrahydrobiopterin dependent aromatic amino acid hydroxylases (AAAHs) comprise a family of enzymes that catalyse the hydroxylation of l‐Phe, l‐Tyr and l‐Trp. These reactions are of central physiological importance. Consequently, dysfunction of the AAAHs is associated with serious disorders, that is, the genetic metabolic disease phenylketonuria for phenylalanine hydroxylase (PAH), and neurological and neuropsychiatric disorders for tyrosine hydroxylase (TH) and tryptophan hydroxylase 1 and 2 (TPH1, TPH2). Mammalian AAAHs are tetrameric proteins that present a three‐domain structure with a remarkable high similarity, in particularly for the catalytic domains. Structural analyses have provided valuable insights on the effect of disease‐associated mutations, mechanism of catalysis, the determinants for substrate specificity and regulation of enzymatic activity. The best characterised AAAH is PAH, which also seems to be the precursor of the family. The AAAHs have developed sophisticated regulatory mechanisms during evolution to control substrate l‐Phe (PAH), catecholamine (TH), serotonin and melatonin (TPHs) levels.

Key Concepts:

  • The aromatic amino acid hydroxylases (AAAHs) are tetrahydrobiopterin (BH4) dependent enzymes that use dioxygen as additional substrate.

  • The AAAHs are involved in serious inborn errors of metabolism with neuronal impact.

  • The AAAHs are highly homologous, contain a catalytic mononuclear nonheme iron coordinated by a 2‐His‐1‐carboxylate facial triad and show similar reaction mechanism.

  • Metazoans have at least three AAAH genes: phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH) and tryptophan hydroxylase(s) (TPH(s)), where PAH appears as the precursor of the family.

  • Mammalian PAH, TH, TPH1 and TPH2 are tetrameric, with a three‐domain subunit structure; substrate specificity is provided by the catalytic domain.

  • The AAAHs are highly regulated by mechanisms at the transcriptional, translational, posttranslational and allosteric levels.

  • Evolutionary adaptation of the enzymes is most evident at the regulatory mechanisms, along the complexity of the organisms, especially of the central nervous system (CNS).

Keywords: tetrahydrobiopterin; mononuclear nonheme iron; 2‐His‐1‐carboxylate facial triad; enzyme regulation; structure–function relationships; inborn errors of metabolism; neurometabolic disorders

Figure 1.

The reactions catalysed by mammalian AAAHs and the synthesis and regeneration of BH4. The BH4‐dependent reaction of the AAAHs leads to the hydroxylation of the respective aromatic amino acid (grey panel) and the conversion of BH4 into BH4–4α‐carbinolamine. The two enzymes pterin 4α‐carbinolamine dehydratase and dihydropteridine reductase (DHPR) are responsible for the regeneration of the cofactor, necessary for a new cycle of hydroxylation (light blue panel). In the absence of DHPR, quinoid dihydrobiopterin can be converted non‐enzymatically to BH2 and reduced to BH4 by dihydrofolate reductase (DHFR) through a salvage pathway (dashed arrows in light blue panel). BH4 is synthesised de novo from guanosine tripshophate (GTP) by the action of three enzymes; GTP cyclohydrolase I, pyruvoyltetrahydropterin synthase and SR (yellow panel). There are a number of alternate enzymes and salvage pathways that also lead to the synthesis of BH4 (dashed arrows in yellow panel).

Figure 2.

Sequence and structure of the AAAHs. (a) Schematic representation of the sequence alignment of human PAH, TH (isoform 1), TPH1 and TPH2. Residues conserved in all four proteins are depicted with a red bar above the alignment scheme. Phosphorylation sites are marked with a yellow star and correspond to positions Ser16 for PAH; Thr8, Ser19, Ser31, Ser40 for TH1; Ser58 for TPH1 and Ser19 for TPH2. (b–d) Structure of the AAAHs subunits coloured yellow (regulatory domain), blue (catalytic domain) and red (oligomerization domain). The cofactor BH4 and the amino acid substrates are shown in orange and yellow spheres, respectively. The solution structure of the regulatory ACT domain of TH is shown in white cartoon (PDB id 2MDA). (e–f) Tetrameric assemblies of PAH and TH generated by combining the quaternary structures of the oligomerization and catalytic domains (PDB id 2PAH for PAH; 1TOH for TH) with the structure of (e) the regulatory and catalytic domains (PDB id 1PHZ for PAH) or (f) the solution structure of dimeric regulatory domain (PDB id 2MDA for TH) in accordance with (Zhang et al., ). Only subunits I and IV are coloured for visualisation purposes. (g) Hypothetical tetrameric model of TPH1. For this enzyme only structural data of the truncated catalytic domain is so far available (shown here: PDB id 3E2T), and the tetramer is, therefore, blurred.

Figure 3.

Binding interactions of the AAAHs. (a) Cofactor binding site of PAH (PDB id 1J8U) with the cofactor BH4 in ball and stick representation (orange) showing stacking interactions with Phe254 and electrostatic interactions with Glu286, which are conserved in all four AAAHs. (b) Substrate binding site of TPH1 (PDB id 3E2T) with bound amino acid substrate (l‐Trp; in yellow) showing the interaction with conserved Arg257. (c) l‐DOPA bound to PAH (PDB id 6PAH); the hydroxyl groups of the catechol replace two iron‐bound water molecules. (d) Ser19‐phosphorylated N‐terminal peptide of TH bound to 14‐3‐3γ (PDB id 4J6S).

Figure 4.

Substrate‐dependent conformational changes in the catalytic domain. (a) Structural comparison of TPH1 with cofactor (PDB id 1MLW) and with substrate (PDB id 3E2T). Cofactor‐ and substrate‐bound structures are shown in white and grey cartoon, respectively. Loops consisting of residues Val122‐Phe136, Gln362 and Asp371 (in TPH1) closing around the active site upon substrate binding are highlighted in red (without substrate) and yellow (with substrate). (b–c) Principal component analysis of the available X‐ray structures of the AAAHs. (b) Visualisation of principal component 1 which consists of a subdomain (coloured red) moving in over the substrate binding site relative to a more rigid core. (c) Available X‐ray structures projected onto the two first principal components with each dot representing one structure of the catalytic domain. The subunits can be divided into two major groups based on their pairwise structural deviations: (i) substrate bound, and (ii) apo and pterin bound, with a group of inhibitor bound structures of TPH1 obtaining an intermediate state.

Figure 5.

AAAHs phylogenetic analysis of representative organisms. Nodes coloured red and blue depict PAH from bacteria and protozoa, respectively. Sequences marked with an asterisk represent unreviewed sequences in the Uniprot database



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Skjærven, Lars, Teigen, Knut, and Martinez, Aurora(Aug 2014) Structure–Function Relationships in the Aromatic Amino Acid Hydroxylases Enzyme Family: Evolutionary Insights. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0025581]