Critical Review
Phenylalanine Hydroxylase: Function, Structure,
and Regulation
Department of Biomedicine and K.G. Jebsen Centre for Research on
Neuropsychiatric Disorders, University of Bergen, Jonas Lies vei 91, 5009-
Bergen, Norway
Abstract
Mammalian phenylalanine hydroxylase (PAH) catalyzes the
rate-limiting step in the phenylalanine catabolism, consum-
ing about 75% of the phenylalanine input from the diet and
protein catabolism under physiological conditions. In
humans, mutations in the PAH gene lead to phenylketonuria
(PKU), and most mutations are mainly associated with PAH
misfolding and instability. The established treatment for PKU
is a phenylalanine-restricted diet and, recently, supplementa-
tion with preparations of the natural tetrahydrobiopterin
cofactor also shows effectiveness for some patients. Since
1997 there has been a significant increase in the understand-
ing of the structure, catalytic mechanism, and regulation of
Keywords: enzymology; evolution; protein function; protein structure
Introduction
Phenylalanine hydroxylase (PAH, EC 1.14.16.1) catalyzes the
conversion of L-phenylalanine (L-Phe) to L-tyrosine (L-Tyr) by
para-hydroxylation of the aromatic side-chain. In mammals,
this tetrahydrobiopterin (BH4)-dependent reaction is the initial
and rate-limiting step in the degradation of excess L-Phe from
dietary proteins, where L-Tyr is further degraded to products
that feed into the citric acid cycle (Fig. 1). L-Tyr is thus a non-
essential amino acid in PAH-containing organisms, where it is
used for protein synthesis or as precursor for neurotrans-
C 2013 International Union of Biochemistry and Molecular Biology, Inc.
V known about the structure–regulation relationships (5). PAH is
Volume 65, Number 4, April 2013, Pages 341–349
*Address for correspondence to: Aurora Martinez, Department of
Biomedicine and K.G. Jebsen Centre for Research on Neuropsychiatric
Disorders, University of Bergen, Jonas Lies vei 91, 5009-Bergen, Norway.
Tel: þ47-55586427. E-mail: aurora.martinez@biomed.uib.no.
Received 4 December 2012; accepted 9 January 2013
DOI: 10.1002/iub.1150
Published online 4 March 2013 in Wiley Online Library
(wileyonlinelibrary.com)
IUBMB Life
Marte I. Flydal
Aurora Martinez*
PAH by its substrate and cofactor, in addition to improved
correlations between genotype and phenotype in PKU.
Importantly, there has also been an increased number of
studies on the structure and function of PAH from bacteria
and lower eukaryote organisms, revealing an additional ana-
bolic role of the enzyme in the synthesis of melanin-like pig-
ments. In this review, we discuss these recent studies,
which contribute to define the evolutionary adaptation of the
PAH structure and function leading to sophisticated regula-
tion for effective catabolic processing of phenylalanine in
mammalian organisms. V C 2013 IUBMB Life, 65(4):341–349,
2013
mitters and other L-Tyr derivatives. The function of PAH in
mammals has been studied since the end of the 1950s (see ref.
1 for a review of the earlier studies). PAH is primarily present
in the liver, where removal of excess L-Phe prevents the neuro-
toxic effect of hyperphenylalaninemia (HPA). However, L-Phe is
also an essential proteinogenic amino acid and it is important
to avoid that it is fully catabolized. To accomplish this dual
role of preserving, yet removing excess L-Phe effectively, mam-
malian PAH has developed several regulatory mechanisms and
a specific structure—the framework through which the regula-
tory properties are exerted. In recent years, there have been
important advances in the elucidation of the catalytic mecha-
nism and structure–function relationships (2–4), but less is
also present in non-mammalian eukaryote organisms and
some bacteria, and notably in the last decade several studies
have investigated PAH from these sources. In addition to cata-
bolic degradation (6,7), PAH from bacteria and non-mamma-
lian eukaryote organisms has a prominent anabolic role con-
tributing to the synthesis of melanin-type pigments (8–11) (Fig.
1). This review focuses on the present understanding of the
structure, function, and regulation of mammalian PAH, and
341
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The PAH reaction and complete L-Phe catabolism (black pathway). In blue (and squared gray background), the abnormal me-
FIG 1 tabolism of L-Phe in phenylketonuria, through transamination and decarboxylation of accumulated L-Phe. In red (and round
grey background), the production of pyomelanin from homogentisate occurring in some bacteria. [Color figure can be viewed
in the online issue, which is available at wileyonlinelibrary.com.]

includes a comparative discussion of the enzyme from different
organisms along an evolutionary perspective.
Phenylketonuria
Dysfunctional PAH leads to increased concentration of L-Phe in
the blood and the appearance in urine of metabolites that arise
from the transamination of L-Phe to phenylpyruvate (Fig. 1).
This is the hallmark of the HPAs, of which classic phenylketonu-
ria (PKU) (OMIM 261600) is the most severe form with plasma L-
Phe levels >1,200 lM (12). The accumulation of L-Phe and the
subsequent disturbance in brain neurotransmitters lead to neu-
rological symptoms including mental retardation, purposeless
movements, and depression (for a complete list of symptoms,
see www.omim.org). The dietary intake of L-Phe must therefore
be strictly controlled in PKU patients (12–14).
PKU is inherited as an autosomal recessive disorder, with
more than 500 disease-causing mutations (www.pahdb.
mcgill.ca and www.biopku.org). In vitro studies of mutant

342 Evolutionary Adaptation of PAH Structure and Function


proteins have revealed the kinetic and conformational defects
caused by the mutations. PKU appears largely as a misfolding
disease where loss of enzymatic function is caused mainly by
folding defects leading to decreased stability, and PKU is con-
sidered a paradigm for misfolding loss-of-function genetic dis-
orders (13,15,16). The molecular basis for the neurological
symptoms is not completely understood, but saturation by
L-Phe of the LAT-1 transporter at the blood–brain barrier
and defective myelination seem critical. Recently, a new
amyloidosis-like etiology for PKU has been suggested, since
L-Phe has been shown to self-assemble into toxic fibrils (17).
While displaying highest affinity for L-Phe, LAT-1 is also
the only route for eight other large neutral amino acids
(LNAAs) into the brain (18). LNAAs are important for cerebral
protein synthesis, and L-Tyr and L-Tryptophan (L-Trp) are also
precursors for neurotransmitters. Saturation of LAT-1 by ele-
vated levels of L-Phe thus creates an imbalance in both neuro-
transmitter- and protein synthesis, including hypomyelination.
Consequently, supplementation with LNAAs is one treatment
strategy currently investigated, in addition to enzymatic therapy
with pegylated phenylalanine ammonia lyase, gene therapy,
pharmacological chaperones and the already approved supple-
mentation with the cofactor BH4 (for a review see 14). A number
of studies have contributed to reveal the most recurrent geno-
types associated with BH4-responsive PKU and the molecular
mechanisms behind the corrective effects of BH4 (12,19).
Aromatic Amino Acid Hydroxylases
PAH is a member of the aromatic amino acid hydroxylase
(AAAH) enzyme family. The AAAHs share a requirement for a
catalytic non-heme ferrous iron, BH4 as cofactor, and molecu-
lar oxygen as additional substrate. The mammalian genes
encode PAH, tyrosine hydroxylase (TH), and two tryptophan
hydroxylases (TPH1 and TPH2), which are named after their
specific amino acid substrates. The products of TH (L-3,4-dihy-
droxyphenylalanine (L-DOPA)) and the TPHs (5-hydroxytrypto-
phan) are precursors for important neurotransmitters and
hormones in the brain and the neuroendocrine system (2). The
AAAHs show high sequence identity (Fig. 2A), similar struc-
ture, and presumed similar catalytic mechanism (2,3). Metazo-
ans have three or four AAAHs-encoding genes, but only a sin-
gle gene has so far been found in protozoans, such as
Leishmania major (20), and in the slime mold Dictyostelium
discoideum (21). These single AAAHs have been identified as
PAHs (20,21). Moreover, PAH is the only AAAH found in bacte-
ria. In fact, based on the homology between the regulatory
AAAH domains and prephenate dehydratase, Gjetting et al.
proposed that bacterial PAH and prephenate dehydratase
were the precursors of multi-domain AAAHs (22). A role of
PAH as the ancestral function in the AAAH family appears rea-
sonable since the TH and TPH functions are likely to be of
increasing importance in pluricellular organisms. As will be
shown in the next sections, PAH has evolved its structural or-
Flydal and Martinez
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ganization to enable sophisticated regulation, thereby adapting
to the emerging metabolic and neurological needs of high-
complexity organisms.
The Phenylalanine Hydroxylase System
As for the other AAAHs, PAH catalyzes the hydroxylation of its
substrate by incorporation of one oxygen atom into the aro-
matic ring, and the final reaction also includes the reduction
of the second oxygen atom to water using two electrons sup-
plied by BH4. The cofactor BH4 functions as a co-substrate
that is also hydroxylated at each turnover to pterin-4a-carbi-
nolamine (4a-OH-BH4), with consequent dissociation from the
enzyme (23) (Fig. 1). The dehydration and reduction of 4a-OH-
BH4 back to BH4 is catalyzed sequentially by pterin
carbinolamine dehydratase, which dehydrates 4a-OH-BH4 to
dihydrobiopterin quinonoid (q-BH2), and the NADH-dependent
dihydropteridine reductase, respectively (23). These two
enzymes are therefore considered as part of the PAH system,
making the degradation of L-Phe sensitive to defects in several
genes. If q-BH2 rearranges to BH2, reduction to BH4 can be
catalyzed by dihydrofolate reductase (23).
Structure of Mammalian PAH
Mammalian PAH is a homo-tetrameric enzyme of 50 kDa sub-
units. The structure of a full-length mammalian PAH has not
yet been solved, but truncated PAH structures are available
(Table 1). A composite model of full-length tetrameric PAH can
be prepared based on crystal structures from dimeric trun-
cated rat PAH (PDB 2PHM) and tetrameric human PAH (hPAH)
(PDB 2PAH) (Fig. 2B). Although only the BH4-responsive PKU
mutant A313T-PAH has been amenable to crystal structure
determination (19), the available structures have been crucial
for correlating genotypes and phenotype in PKU, defining hot-
spots for enzyme destabilization (4,15).
Each PAH subunit is composed of an N-terminal regula-
tory domain (residues 1–110), a central catalytic domain
(residues 111–410), and a C-terminal oligomerization domain
(residues 411–452) (24,25) (Fig. 2B). The N-terminal regula-
tory domain is classified as an ACT-domain, a regulatory mod-
ule present in several proteins, which often dimerizes and
binds amino acids (26). This domain is flexibly attached to the
catalytic domain via a hinge region (Arg111–Thr117), but
establishes extensive contacts with the catalytic domain of the
adjacent subunit within the dimer. The regulatory domain is
necessary for manifestation of the regulatory properties, such
as activation by L-Phe, and it is still debated whether or not it
includes an allosteric binding-site for L-Phe. In support of the
existence of a regulatory site, Gjetting et al. remarked that the
regulatory domain presents two motifs, GAL (residues 46–48
in hPAH) and (I/L)ESRP (residues 65–69), which are involved
in L-Phe binding in a bacterial prephenate dehydratase, and
demonstrated that isolated regulatory domains of hPAH
343
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IUBMB LIFE

Domain organization and structure of PAH. A: Alignment of human AAAHs and PAHs from different organisms. Bars represent
FIG 2 gaps (white), non-identical residues (gray), residues identical in
60% of the sequences (dark gray), and residues identical in
all the sequences (black). RD, regulatory domain; CD, catalytic domain; OD, oligomerization domain. The highest homology
(80%) is encountered in the catalytic domains. B: Composite model of full-length tetrameric human PAH prepared with PDBs
2PHM and 2PAH. Inset, domain organization of each subunit. C: The structure of the ternary PAH  Fe(II)  BH4  L-Phe complex,
based on PDB 1MMK, with L-Phe modeled at the 3-(2-thienyl)-L-alanine binding site. D: The crystal structure of PAH from
C. violaceum PAH (PDB 1LTV). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

actually form a dimer and bind L-Phe (22). However, whether
this intersubunit regulatory binding site is functional in full-
length PAH, where (according to the available structural mod-
els) regulatory domains are physically separated (Fig. 2B), has
not been convincingly demonstrated (see also next section).
Preceding the ACT-domain is an intrinsic autoregulatory
sequence (residues 1–33 in hPAH) that extends over the active
site (24) and limits L-Phe access, especially when its physiologi-
cal cofactor (6R)-BH4 is bound (24,27–29). The highly mobile
N-terminal (residues 1–18) of the intrinsic autoregulatory
sequence is not seen in the crystal structure, and conforma-
tional information has been obtained by molecular modeling
(30).
The catalytic domain contains the binding sites for iron,
cofactor, and substrate. At the active site iron binds to two his-
tidines (His285 and His290 in hPAH) and a glutamate (Glu330
in hPAH) (Fig. 2C). Structures with bound L-Phe analogues
3-(2-thienyl)-L-alanine or L-norleucine, and reduced or oxi-
dized cofactor at the active site (Table 1; Fig. 2C) have
provided the molecular frames to elucidate the AAAH mecha-
nism (2,3,31). Formation of the high-spin Fe(IV) hydroxylating
species—identified experimentally in the catalytic cycles of
both TH (32) and PAH from Chromobacterium violaceum
(33)—is a crucial step in the reaction.
The oligomerization domain starts by an antiparallel-sheet
(residues 411–414, 421–424), responsible for dimerization, fol-
lowed by a 40 Å long-helix (428–452) that mediates tetrameri-
zation through domain swapping and antiparallel coiled-coil
formation with the other monomers (25) (Fig. 2B).
Regulation of Mammalian PAH by L-Phe,
BH4, and Phosphorylation
Several mechanisms act together to tightly control the activity of
mammalian PAH, and some short-term effects known to play a
physiological role are caused by L-Phe, the natural cofactor (6R)-
BH4, and phosphorylation at Ser16 by cAMP-dependent protein
kinase. Liver PAH from different mammals displays the same
physical and regulatory properties as the human enzyme,

344 Evolutionary Adaptation of PAH Structure and Function

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Representative PAH structures solved by crystallography
TABLE 1

PDB ID Domains (residues) State Ligands Organism

1PAH CD (117–427) D H. sapiens

2PAH CD, OD (118–452) T H. sapiens
3PAH CD (103–427) D L-adrenaline H. sapiens
4PAH CD (103–427) D L-noradrenaline H.sapiens
5PAH CD (103–427) D L-dopamine H. sapiens
2PHMa RD, CD(1–429) D R. norvegicus
a,b
1PHZ RD, CD(1–429) D R. norvegicus
1DMW CD (118–424) D BH2 H. sapiens
1J8U CD (103–427) D BH4 H. sapiens
1LTV CD (1–275) M C. violaceum
1LTZ CD (1–275) M BH2 C. violaceum
1MMK CD (103–427) D BH4, 3-(2-thienyl)-L-alanine H. sapiens
1MMT CD (103-427) D BH4, L-norleucine H. sapiens
1TG2 CD A313T (103-427) D BH2 H. sapiens
2V27 CD (1-275) M C. psychrerythraea

RD, regulatory domain; CD, catalytic domain; OD, oligomerization domain; D, dimeric; T, tetrameric; M, monomeric.
a
No structural information for residues 1–18.
b
Phosphorylated at Ser16.

although there are some differences in the activation constants
and notably the extent of the activation by L-Phe (5).
The activity of tetrameric mammalian PAH—when assayed
with 6R-BH4—is activated by incubation with L-Phe and
responds with positive cooperativity toward increasing sub-
strate concentrations (Hill coefficient 2) (1,34). The need for
fine-tuned conformational changes to elicit an allosteric
response by L-Phe is manifested by the loss of positive coopera-
tivity in many PKU-associated hPAH mutants that still maintain
the tetrameric structure (19). Current models to explain alloste-
ric regulation with positive cooperative ligand-binding describe
the transition from a tense (T), less active state with low affinity
for the ligand, to a relaxed (R), more active, high-affinity state. A
large conformational transition is indeed visualized for mam-
malian PAH upon activation by substrate as shown among other
by the surfacing of a buried tryptophan residue and an increase
in hydrophobicity and size (1,5,29,34). The mechanism of this
activation is not yet understood and there is no consensus in the
field as to whether the activation is caused by L-Phe binding to
an allosteric site in the regulatory domain (22,35,36) or to the
active site itself (37–39). The numerous studies related to this
subject have been recently reviewed by Fitzpatrick (5). The first
indication that binding to a regulatory site might be unnecessary
to generate positive cooperative response arose when it was
shown that noradrenaline, which binds directly to the ferric iron
in the active site, also elicited positive cooperativity (37). Later,
differential scanning calorimetry investigations further sup-
ported that L-Phe only binds to the catalytic domain of hPAH
(38). Moreover, surface plasmon resonance analyses and site-
directed mutagenesis on crystallographically defined hinges
have also identified the active site as the epicenter of the global
conformational changes resulting in activation and positive
cooperativity observed in the full-length tetrameric enzyme
(29). Nevertheless, evidences for L-Phe binding by the isolated
regulatory domains have been put forward by isothermal titra-
tion calorimetry (22) and nuclear magnetic resonance spectros-
copy (36). Interestingly, recent analyses reveal that substrat
binding sites in fact exist in the regulatory ACT domain in hPAH,
but binding appears obstructed by residues from the catalytic
domain (39). It seems therefore not surprising, neither contra-
dictory, that L-Phe binds to isolated ACT domains (22,36).
The cofactor BH4 also exerts a regulatory inhibitory effect
on mammalian PAH, manifested as a stabilization of the
T-state, which is reverted by L-Phe activation (1,5,27). The

Flydal and Martinez 345


negative effect on activity is attributed to the intrinsic autoreg-
ulatory sequence (28), and molecular dynamics (MD) simula-
tions complemented with thermodynamic characterizations of
BH4-binding also support a restructuring of the intrinsic auto-
regulatory sequence, which possibly occupies the L-Phe binding
site (27). In the liver, PAH exists largely in this BH4-bound,
low-activity, stable state (40). The stabilizing effect of BH4 is
one of the most important molecular mechanisms behind the
stimulation of PKU-mutant activity by BH4 supplementation
(i.e., the chaperone effect) (19).
Phosphorylation of mammalian PAH at Ser16 by glucagon-
stimulated cAMP-dependent protein kinase increases the affin-
ity for L-Phe and thus activates the enzyme in synergy with
L-Phe activation (1,5). Crystal structures of unphosphorylated
and phosphorylated forms of dimeric rat PAH (24) (Table 1)
lack structural information on the region around Ser16. Site-
directed mutagenesis and molecular modeling indicate that
phosphorylation elicits local conformational changes, mostly
driven by electrostatics, that increase the accessibility of the
substrate binding site (30).
PAH in Non-Mammalian Eukaryote
Organisms
The investigation of PAH in non-mammalian organisms includ-
ing bacteria readily followed the earliest studies of mammalian
PAH (41). Recently, PAH has also been identified in some pro-
tozoans and slime molds, and even in nonflowering plants
(20,21,42). While animal PAH uses BH4 as cofactor, other nat-
ural tetrahydropterins, such as tetrahydrodictyopterin, are
present in D. discoideum together with BH4 and support the
PAH function at least in vitro (21,43). However, BH4 seems to
be the PAH cofactor in vivo, while tetrahydrodictyopterin func-
tions mainly as an antioxidant (43). Plant PAH seems to use
tetrahydrofolate as cofactor (42).
In addition to their role in the catabolic L-Phe/L-Tyr degra-
dation pathway (7), PAH in lower eukaryotes has been impli-
cated in the synthesis of (pyo)melanin, a well-known pigment
that confers advantageous properties (8–10,20). Melanin is
used by insects to encapsulate parasites (9) and by the sponge
Geodia cydonium to target cells for apoptosis (8). In the nema-
tode Caenorhabditis elegans, PAH is expressed in the hypoder-
mal cells, and knock-out pah mutants lack a melanin in the
cuticle which appears to protect from oxygen-radicals (10).
Bacterial PAH
In bacteria, the gene coding for PAH (phhA) occurs within sev-
eral phyla, but appears to be relatively scattered within these.
The phylogenetic distribution of phhA in bacteria has not been
investigated per se, but Pribat et al. report that 184 out of 724
bacterial genomes encode a PAH (44). In some PAH-containing
bacteria, phhA is located next to phhB, encoding pterin carbi-
346
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nolamine dehydratase, in the phh operon, which also includes
the gene for an aromatic aminotransferase (45). The similarity
of the regulatory domain of mammalian PAH with pterin car-
binolamine dehydratase has been remarked, and the bacterial
operon-structure has been proposed as a possible basis for
evolution of the modular gene encoding eukaryotic PAH (24).
Several bacterial PAHs have been studied experimentally
to different extents, all showing an absolute requirement for
ferrous iron and tetrahydropterin (11,45–47). It has been
speculated that most bacterial PAHs use alternative pterins to
BH4, for example, tetrahydromonapterin (44). For the Colwel-
lia psychrerythraea enzyme, highest activity was however
measured with BH4 (47). Except for the recently studied PAH
from Legionella pneumophila, which appears to be dimeric
(11), presently characterized bacterial PAHs are monomeric
and show a similar fold to the catalytic domain of mammalian
PAH (46,47) (Table 1; Fig. 2D).
Several pathogens encode a PAH and produce a melanin-
pigment, notably a pyomelanin derived from homogentisate
autooxidation and polymerization (Fig. 1). The high mutational
rate of Pseudomonas aeruginosa during the course of lung
infection leads to adaptive mutations in the gene encoding
homogentisate oxidase (Fig. 1) (48). This induces increased
homogentisate accumulation and hyperproduction of pyomela-
nin that render the infection very difficult to treat (48). The
pyomelanin of the opportunistic pathogen Burkholderia ceno-
cepacia was recently shown to have antioxidant properties,
and mutants not producing pyomelanin were more sensitive to
oxidative stress (49). Furthermore, the crucial role of L. pneu-
mophila PAH in both the growth of the bacterium in tyrosine-
limited media and in the synthesis of pyomelanin has been
recently demonstrated (11) (Fig. 3). The secreted pyomelanin
has intrinsic ferric reductase activity, contributing to the
capacity of Legionella to acquire iron and possibly to its viru-
lence (50).
Adaptive Strategies: Regulation Of PAH
Activity By L-Phe
PAH shows an evolutionary adaptation of its structure to
accommodate for changes in the regulation of its function. The
characterization of PAHs in organisms at different levels of
cellular complexity indicates that the tetrameric structure
appears early—in an evolutionary scale—in the animal king-
dom (21). Clear manifestations of allosteric regulation of PAH
activity by L-Phe have only been proven for mammals, but the
need for PAH activity in anabolic/catabolic pathways with si-
multaneous preservation of threshold levels of L-Phe for pro-
tein synthesis would require a strict enzyme regulation in all
organisms. Several mechanisms appear to contribute to main-
tain L-Phe homeostasis in different organisms, and, in addition
to the regulation by positive cooperativity in mammals, other
mechanisms such as non-catalytic binding of L-Phe, and low
Evolutionary Adaptation of PAH Structure and Function

Role of PAH in growth and pigment-synthesis in Legionella pneumophila 130b. Wild type 130b and mutant L. pneumophila
FIG 3 lacking PAH (NU406) with empty vector (pMMB2002) or vector containing the pah gene (pPhhA) were grown in chemically
defined medium (CDM) lacking tyrosine (A) and in medium containing standard (CDM) (B,C) and twice the standard (CDM^ 2
Tyr) amount of tyrosine (B). The importance of PAH both for growth in media limited in tyrosine (A), and for the production of
pyomelanin pigment (B,C) was established. Figure adapted from ref. 11. [Color figure can be viewed in the online issue, which
is available at wileyonlinelibrary.com.]
substrate affinity have been revealed in recent in vitro studies
with purified PAHs from C. elegans and bacteria.
PAH from C. elegans, which is tetrameric and includes the
canonical three-domain PAH organization and 57% sequence
identity with hPAH, does not show positive cooperativity (10).
This sophisticated regulatory mechanism that allows mamma-
lian PAH to avoid toxic accumulation of L-Phe is not present in
the worm most probably because it is simply not required at
its level of nervous system complexity. In fact, knock-out pah
worms appear healthy even when grown in media supple-
mented with excess L-Phe (10), suggesting that accumulation
of L-Phe is not detrimental to the C. elegans nervous system.
Nevertheless, it is reasonable to infer that the worm would
face the need of maintaining threshold values of L-Phe for protein
synthesis. In this context, our discovery of a second binding site for
L-Phe in the regulatory domain of worm PAH (39) suggests a sim-
pler regulatory mechanism where the non-catalytic binding of L-
Phe, which does not seem to induce activating conformational
changes, rather ensures the preservation of certain L-Phe levels.
In the case of bacteria, the cold-adapted PAH from C. psy-
chrerythraea shows a low affinity for L-Phe, and the concen-
tration of substrate providing half maximal activity ([S]0.5, a
parameter used in non-Michaelis–Menten kinetics to provide a
constant comparable to Km) is 1300 6 300 lM, much higher
than [S]0.5 < 200 lM for eukaryote PAH (1,10). This could be a
result of the increased flexibility and accessibility of the active
site due to cold adaptation, but might also indicate a primitive
regulatory mechanism to preserve threshold amounts of L-Phe.
Indeed, also the thermostable PAH from L. pneumophila dis-
played low affinity for L-Phe (Km ¼ 735 6 50 lM). But not all
bacterial PAHs seem to have low affinity for its substrate and
PAH from C. violaceum has Km (L-Phe) comparable to eukar-
yote PAH (51). Additional studies are necessary to reveal the
extent of low affinity for L-Phe among bacterial PAH. In fact
other mechanisms at the transcriptional level, such as the reg-
ulation of transcription from phhA by L-Phe shown to take
place in Pseudomonas putida (52), might also be important to
safeguard L-Phe (and aromatic amino acid) homeostasis.
Flydal and Martinez
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Evolutionary Change in the Function of
the PAH-Catalyzed Reaction
It is broadly accepted that the main role of mammalian PAH is
the catabolic degradation of dietary L-Phe and protection from
HPA. On the other hand, combining the studies from Calvo
et al. and Fisher et al. shows that the role of C. elegans PAH is
catabolic, but also anabolic, providing L-Phe for melanin syn-
thesis (7,10). In fact, functional divergence analysis acknowl-
edge that an important functional switch has occurred
between the nematode PAH and mammalian PAH on the evo-
lutionary time-scale (39). These results further indicate that
the specific residue substitutions are associated to fundamen-
tal changes in regulation of the activity (10). The more sophis-
ticated regulation in the mammalian organisms seems to tune
the enzyme toward the effective catabolic processing of L-Phe.
Acknowledgements
Authors are very thankful to all members of the Biorecognition
research group for discussions, especially to Jarl Underhaug who
also contributed to the figures. This research is supported by
grants from the Research Council of Norway, the Western Nor-
way Health Authorities, K.G. Jebsen Centre for Research on Neu-
ropsychiatric Disorders and Novo Seeds (Novo Nordisk Fonden).
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