MINIREVIEW
Evolutionary changes to transthyretin: structure and
function of a transthyretin-like ancestral protein
Sarah C. Hennebry
Department of Biochemistry and Molecular Biology, Bio21 Institute, The University of Melbourne, Victoria, Australia
Introduction
The evolution of the structure and the function of the
thyroid hormone (TH) distributor, transthyretin, has
been well researched. The primary, secondary, tertiary
and quaternary structures of this vertebrate protein are
highly conserved. It was therefore hypothesized that
the transthyretin gene may have evolved in a nonverte-
brate organism. Searches for a transthyretin progenitor
led to the identification of a transthyretin homolog,
which was found initially in nonvertebrate genomes
and subsequently in all major kingdoms. The evolution
of the structure and function of the transthyretin
homolog [referred to as transthyretin-like protein
(TLP)] has been the focus of recent studies by several
research groups. TLPs from various organisms have
been demonstrated to share remarkable structural
similarities to vertebrate transthyretins. Despite this
Keywords
evolution; purines; structure; transthyretin;
transthyretin-like protein
Correspondence
S. C. Hennebry, Human Neurotransmitters
Laboratory, Baker IDI Heart and Diabetes
Institute, P.O. Box 6492, St Kilda Road
Central Melbourne, Victoria 3008, Australia
Fax: +61 3 8532 1100

sequence; RNAi, RNA interference; TH, thyroid hormone; TLP, transthyretin-like protein.
FEBS Journal 276 (2009) 5367–5379 ª 2009 The Authors Journal compilation ª 2009 FEBS 5367
structural similarity, TLP and transthyretin have dif-
ferent functions. TLP is an enzyme functioning in the
purine catabolism pathway, where it hydrolyses 5-hy-
droxyisourate (5-HIU), the oxidation product of uric
acid. Phylogenetic analyses have revealed that it is
likely that the transthyretin gene arose as a result of a
duplication of the TLP gene in early vertebrate evolu-
tion. Thus, the evolution of TLP and transthyretin rep-
resents a remarkable case of the divergent evolution
from an enzyme to a hormone distributor.
This minireview will present and discuss recent find-
ings regarding the identification and distribution of
TLP genes in nature, the structural and functional
characterization of the TLP from various organisms,
and the evolution of TLP and transthyretin.
The identification of TLPs and
transthyretins in nature
The evolution of transthyretin and its distribution in
nature have been well researched [1,2]. Several studies
have demonstrated that all vertebrates synthesize
transthyretin at some stage during their development
[2–4] and this synthesis is primarily localized to the
liver, choroid plexus and retinal pigment epithelium.
The expression of the transthyretin gene in vertebrates
occurs independently in these tissues [5,6]. There is
considerable sequence identity and similarity between
the amino acid sequences of transthyretin from various
vertebrate organisms. The most divergent transthyretin

genomes to mine, Eneqvist et al. [10] used bla st searches
to identify a further 49 putative TLP sequences in the
genomes of bacteria, plants and invertebrate animals.
The TLP genes they identified typically encoded a pro-
tein of 114 amino acid residues compared with, on
average, 125 residues in transthyretin (the number of
residues was species dependent). Furthermore, Eneq-
vist et al. [10] observed that all TLP sequences
possessed a consensus C-terminal tetrapeptide: Tyr-
Arg-Gly-Ser. Alignment of TLP and transthyretin
sequences revealed that the regions of greatest similar-
ity between the two families of proteins were in the
N-terminal and C-terminal regions [11]. In order to
distinguish between the two protein families in greater
detail, a comparative analysis of TLP and transthy-
retin sequences was performed [11]. In this study, a set
of bacterial TLP and vertebrate transthyretin
sequences was probed for motifs that might be con-
served in each group. The study revealed that the
transthyretin sequences in this set were so similar that
a single motif spanned the entire length of each protein
sequence. However, in the set of TLP sequences, five
specific motifs were identified, namely motifs A–E
(with motif A being the most highly conserved). The
motifs in the TLP sequences were found in the follow-
ing arrangement (from N-terminal to C-terminal):
(E)-B-D-C-A (see Fig. 1A). Motif E was only found in
TLPs from plant species and from two alphaproteo-
bacteria: Bradyrhizobium japonicum and Magnetospiril-
lum magnetotacticum. Motif E is homologous to

Interestingly, whilst motifs A’–C’ represent the
regions of greatest sequence similarity between TLP
and transthyretin, they also contain specific amino acid
substitutions that enabled the distinction of one group
from the other. For instance, at their C-termini (motif
A’ region), nearly all TLP sequences possess a Tyr-
Arg-Gly-Ser tetrapeptide. Specifically, the tyrosine and
glycine residues were found to be 100% conserved
among TLP sequences. Upon sequence alignment with
TLP, the residues at the same positions in transthyretin
are threonine and valine, respectively. At the N-termini
of TLP sequences (the motif B’ region) a conserved his-
tidine residue was found. The equivalent residue in
transthyretin sequences is lysine (also 100% conserved).
Interestingly, the residues involved in TH binding in
transthyretin are not conserved in TLP sequences.
Rather, it appears that residues involved in the struc-
tural integrity of the TLP ⁄ transthyretin molecule have
been conserved. The alignment of representative trans-
thyretin and TLP sequences in Fig. 2 demonstrates the
distribution of residues that are 100% conserved in
both TLP and transthyretin sequences as well as those
that are 100% conserved solely within the set of TLP
sequences.
Distribution of TLPs and transthyretins
in nature
The distribution of TLP in nature and its evolutionary
relationship to transthyretin have been studied exten-
sively in recent years [10,11]. To date, TLP genes have
been identified in over 200 organisms across all king-

sea bream transthyretin. Motif A’ lines the
hydrophobic core. Motif B’ forms the
dimer–dimer interface and the opening of
the central channel of the TTR molecule.
Residues in motif C’ are involved in mono-
mer–monomer interactions. (Modified from
[11]).
S. C. Hennebry The evolution of the transthyretin-like protein
FEBS Journal 276 (2009) 5367–5379 ª 2009 The Authors Journal compilation ª 2009 FEBS 5369
doms. By contrast, the transthyretin gene is only found
in vertebrates. Whilst the TLP gene is widely distributed
in nature, there are some notable absences or apparent
‘losses’ of the TLP gene. For instance, no protozoans to
date have been found to have a TLP gene, even though
related organisms such as the slime mold Dictyosteli-
um discoideum and the jakobite Jakoba bahemiensis
both express the TLP gene. A TLP gene is absent from
the cnidarian and ascidian phyla, despite the fact that
organisms before and after these branch points in evo-
lution express the TLP gene. This evidence suggests that
whilst TLP might have been conserved throughout evo-
lution because they have an important functional role,
it is by no means essential to all organisms.
Subcellular localization of TLP in
bacteria
In most instances, the TLP gene is present as a single
copy in the organisms in which it has been identified.
The gene typically encodes a cytoplasmic protein and,
in the case of bacteria, is typically located in purine
metabolism operons, neighbouring the gene which

labelled A–H. A single a-helix is indicated with a rectangle. The residues that are strongly conserved between transthyretins and TLPs are
indicated with an asterisk (*). Residues 100% conserved among all TLP sequences are indicated with a hash (#). Numbering for human
transthyretin is shown directly beneath the alignment.
The evolution of the transthyretin-like protein S. C. Hennebry
5370 FEBS Journal 276 (2009) 5367–5379 ª 2009 The Authors Journal compilation ª 2009 FEBS
identified. This N-terminal extension contains a nona-
peptide, which is predicted to encode a type-two
peroxisomal sequence (PTS2) [11,12]. Recently, a
proteomic analysis of leaf peroxisomes confirmed the
peroxisomal localization of the Arabidopsis thaliana
TLP [13]. Found in all eukaryotic cells, peroxisomes
are specialized organelles in which oxidative reactions,
such as those associated with purine metabolism, are
compartmentalized. The co-localization of purine-
metabolism enzymes (e.g. uricase) with TLP in peroxi-
somes is therefore in keeping with the function of the
A. thaliana TLP hydrolysis of the purine 5-HIU (S. C.
Hennebry, unpublished results). These observations
contradict those made by Nam and Li [14], where the
A. thaliana TLP was reported to be localized only in
the cytosol and was unlikely to have a function in pur-
ine metabolism. In this study, the authors failed to
take into account that the A. thaliana gene At5g58220
encoded two distinct proteins: OHCU decarboxylase
and TLP. Therefore, conclusions drawn from yeast
two-hybrid studies were based on interactions of the
N-terminal region of OHCU decarboxylase with the
receptor kinase brassinosteroid-insenitive-1, rather than
interactions made by TLP. Furthermore, their conclu-
sion that the TLP could not be peroxisomal was

have been identified in all kingdoms. Given their high
degree of sequence similarity, it has been hypothesized
that the transthyretin gene arose as a result of a dupli-
cation of the TLP gene at some stage in early verte-
brate evolution [11]. Initial phylogenetic analyses of
TLP and transthyretin sequences showed a branching
of transthyretin slightly before the separation of the
chordates [17]. Subsequent analyses using the recently
determined transthyretin sequences from lamprey and
recent additions to echinoderm expressed sequence tag
(EST) databases, suggest that the TLP gene duplica-
tion probably occurred just after the separation of
echinoderms (S. C. Hennebry, unpublished results).
Table 1. Bacteria with multiple copies of TLP genes.
Organism Taxonomy (phylum, class)
Genes encoding
cytoplasmic TLP
Genes encoding
periplasmic TLP
Rhodococcus Actinobacteria, Actinobacteria 2 0
Bradyrhizobium sp. Proteobacteria, Alphaproteobacteria 2 0
Sinorhizobium meliloti Proteobacteria, Alphaproteobacteria 2 0
Dinoroseobacter shibae DFL 12 Proteobacteria, Alphaproteobacteria 2 0
Loktanella vestfoldensis SKA53 Proteobacteria, Alphaproteobacteria 2 0
Roseovarius sp. HTCC2601 Proteobacteria, Alphaproteobacteria 2 0
Ralstonia eutropha H16 Proteobacteria, Betaproteobacteria 2 1
Comamonas testeroni KF-1 Proteobacteria, Betaproteobacteria 2 1
Klebsiella pneumoniae Kp342 Proteobacteria, Gammaproteobacteria 1 1
Salmonella enterica ssp. I choloraesuis Proteobacteria, Gammaproteobacteria 0 2
Chromohalobacter salexigens DSM3034 Proteobacteria, Gammaproteobacteria 1 1

into a possible phenotype was not performed. For
example, the worms were not subjected to any type of
environmental stress. In addition, RNAi was per-
formed using dsRNA for a single TLP gene at a time.
As such, the RNAi studies in C. elegans may have
been more informative had double-knockdown studies
been performed.
A role for TLP in purine metabolism was first pro-
posed in 2001. In an effort to develop a greater under-
standing of purine metabolism in the Gram-positive
bacterium, Bacillus subtilis, Schultz et al. [18] generated
a series of insertion mutants. One of these mutations
was made in the TLP gene (pucM), which is located
immediately downstream of the gene encoding uricase.
The bacteria harbouring this mutation were character-
ized as having a reduced rate of proliferation (com-
pared with wild-type bacteria) on media containing
uric acid as the principal source of nitrogen [18].
Purines are major components of nucleic acids and
nucleotides. Subsequently, de novo and salvage path-
ways for purine biosynthesis are important compo-
nents in the metabolism of all organisms. The ability
to degrade purine compounds, either aerobically or
anaerobically, has been identified in all kingdoms [19].
The aerobic degradation of purines is dependent on
the oxidation of hypoxanthine and xanthine to uric
acid via xanthine dehydrogenase ⁄ oxidase (E.C.
1.1.1.204 ⁄ E.C. 1.1.3.22). In humans, anthropoid apes,
birds, uricotelic reptiles and most insects, uric acid is
the end product of purine metabolism and is thus

quently found in close proximity to the uricase gene and
to another gene encoding proteins belonging to COG
3195. In 2005, Lee et al. [28] revealed the ability of
recombinant TLP from B. subtilis and E. coli to specifi-
cally hydrolyse 5-HIU. Importantly, they demonstrated
the inability of human transthyretin to hydrolyse the
same compound. Ramazzina et al. [12] subsequently
showed that mouse TLP hydrolysed 5-HIU and that the
COG 3195 proteins were responsible for the decarboxyl-
ation of OHCU to (S)-allantoin. Thus, the pathway of
the conversion of uric acid to (S)-allantoin via the three
enzymes uricase, TLP (5-HIUase) and OHCU decar-
boxylase was revealed (see Fig. 3). Whether the three
proteins are able to form a multi-enzyme complex
remains to be determined. One could speculate that the
ability to do so would be favourable given the rapid
kinetics of spontaneous decomposition of both 5-HIU
and OHCU.
The evolution of the transthyretin-like protein S. C. Hennebry
5372 FEBS Journal 276 (2009) 5367–5379 ª 2009 The Authors Journal compilation ª 2009 FEBS
To date, the TLP from three bacteria [28–30], one
plant (A. thaliana; S. C. Hennebry, unpublished
results) and two vertebrate species [12,17], have been
analysed for 5-HIU hydrolytic activity and have all
been shown to be 5-HIU hydrolases. Thus, a role for
TLP in this purine degradation pathway is evident
throughout evolution. In addition, the expression of
the TLP gene in some organisms may be uric acid-
dependent. For example, in the Gram-positive bacte-
rium Deinococcus radiodurans, both the uricase and

with varying affinities [32]. TLP does not appear to
share the classic sequence characteristics of cyclic
amidohydrolases (S. C. Hennebry, unpublished
results). Whilst the E. coli TLP was crystallized in the
presence of Zn
2+
, it has been shown that TLP is not a
zinc-dependent hydrolase [17].
Structural comparison of Transthyretin
and TLP
The 3D structures of transthyretin from various organ-
isms have been well characterized. The first transthyre-
tin crystal structure to be solved (that of human) was
published in 1978 [34]. The Protein Database (http://
www.pdb.org) contains multiple crystal structure coor-
dinates for human transthyretin (including multiple
amyloidogenic forms and with various ligands bound).
The crystal structures of transthyretin from rat [35],
chicken [36] and sea bream [37,38] have also been
solved. All of these structures demonstrate the remark-
able conservation of the prealbumin-like fold (as
described by SCOP, ),
which consists of an eight-stranded b-sandwich (strands
A-H) with each sheet adopting a greek-key topology. A
two-turn a-helix usually (with the exception of chicken
transthyretin) exists between strands E and F in trans-
thyretin. The two transthyretin dimers associate, via
nonpolar interactions, between the loops joining stands
G and H with the loops joining strands A and B, mak-
ing the transthyretin tetramer a ‘dimer of dimers.’

˚
[30].
The main differences between the structures of TLP
and transthyretins are found in the loop connecting
b-strands B and C, which is highly exposed to the
solvent in TLP [17]. Interruptions in the b-strands A, G
and H are also observed in TLP structures as a result
of alterations to the formation of hydrogen bonds
between strands. The carbonyls of residues V104 and
P105 (zebrafish TLP numbering), in the middle of
b-strand G, do not form hydrogen bonds with the nitro-
gen atoms of H12 and Y116 of b-strand H in TLP.
The P105 residue, mainly responsible for the b-strand
irregularities, is invariant in TLP sequences, suggesting
a crucial role for the particular conformation observed
in b-strands A, G and H [17].
Structural nature of the TLP and
transthyretin active sites
One of the striking features of transthyretin is the cen-
tral channel of the protein into which the THs bind.
This central channel traverses the entire tetramer. It
has previously been postulated [40] and demonstrated
[7,41] that the characteristics of the N-termini of trans-
thyretin from different organisms account for differ-
ences in the affinity of the two main THs (T3 and T4)
to the channel by hindering or allowing greater accessi-
bility.
The central channel is also present in TLP, albeit
with quite different structural properties. Previously, it
was demonstrated that the regions of greatest similar-

need for a positively charged residue at this site. Sub-
stitution of H105 and Y118 also significantly reduced
enzyme activity, by approximately 90% [30]. Deletion
of the C-terminus tetrapeptide Tyr-Arg-Gly-Ser signifi-
cantly affected enzyme activity, but it has been sug-
gested that S121 does not influence the reaction [29].
Fig. 4. Comparison of the tertiary structure
of TLP with transthyretin. Stereo diagram
showing a superimposition of tetramers of
Salmonella dublin TLP (magenta) with trans-
thyretin from human (1F41, cyan), rat (1KGI,
yellow), chicken (1TFP, orange) and sea
bream (1SNO, green). Tetramers were
superimposed using the A chain only.
(Adapted from [30]).
The evolution of the transthyretin-like protein S. C. Hennebry
5374 FEBS Journal 276 (2009) 5367–5379 ª 2009 The Authors Journal compilation ª 2009 FEBS
Interestingly, those residues playing a role in enzyme
activity are 100% conserved in all TLP sequences and
100% substituted in transthyretin (see Table 2). How-
ever, substitutions at position 121 (to threonine or glu-
tamate) have been observed. None of the mutations
affected the tetrameric assembly of the TLP molecule
[30]. Furthermore, the surface charge of the TLP active
site is considerably different from the equivalent region
in transthyretin [29,30]. An electrostatically positive
groove in TLP contrasts the negatively charged
TH-binding site in transthyretin (see Fig. 5C).
In summary, a comparison of the catalytic cavity of
TLP with the equivalent region of transthyretin (the

TLP is depicted at 90° to those depicted for
transthyretin and TLP in part A. (C) (i) Elec-
trostatic surface potential of human trans-
thyretin with thyroxine bound inside the
negatively charged and deep channel at the
dimer–dimer interface of the protein. (ii) The
equivalent region in TLP is shallow and
positively charged. (Adapted from [30]).
Table 2. Site-directed mutagenesis of conserved residues in TLP.
Transthyretin
residue
Equivalent
residue in TLP
(S. dublin TLP
numbering)
Effect of mutation
on TLP activity Publication
Lys15 His6 Abolishes [30]
Ser52 Asp42 Reduces by 50% [17]
Glu53 Arg44 Abolishes [29]
Thr106 His95 Reduces by 90% [30]
Thr119 Tyr108 Reduces by 90% [30]
Val122 Ser111 No effect [29]
S. C. Hennebry The evolution of the transthyretin-like protein
FEBS Journal 276 (2009) 5367–5379 ª 2009 The Authors Journal compilation ª 2009 FEBS 5375
the central channel of the transthyretin molecule,
allowing for the binding of bulkier ligands such as
THs. Superimposition of the dimer–dimer interface of
TLP with that of transthyretin illustrates the evolu-
tionary changes that resulted in the functional transi-

metazoan and plant species, and the co-regulation of
TLP genes in some bacteria, suggests a concerted
effort in the rapid generation of allantoin. The co-dis-
tribution of uricase, TLP and OHCU decarboxylase
genes in nature reveals that whenever an organism is
found to have a uricase gene, it always has both TLP
and OHCU decarboxylase genes [12]. In vertebrates,
the loss of these three genes through evolution is mir-
rored. For instance, hominoids lost their ability to
degrade uric acid as the result of the inactivation of
the uricase gene in a primate ancestor, some 15 Ma
[42]. In humans, the TLP gene has several inactivating
mutations and the OHCU decarboxylase gene does
not appear to be expressed [12].
Uric acid is a potent antioxidant in biological sys-
tems. Despite uric acid being the end point of purine
metabolism in humans and birds, high levels of allan-
toin have been detected in their plasma [43,44]. Uric
acid chelates transition metal ions (minimizing metal-
catalysed oxidation), scavenges hypochlorous acid, is a
potent quencher of peroxynitrite and reduces haemo-
globin oxidation by nitrite (for a review, see [45]). It
has been suggested that in humans and birds, the
allantoin generated in these organisms could be a mea-
sure of the levels of oxidative stress [44].
The nonenzymatic oxidation of uric acid generates
5-HIU, just as in the uricase reaction. As previously
discussed, 5-HIU is a highly reactive compound,
which, if left to spontaneously decompose, is capable
of forming numerous free-radical species, which ulti-

TLP: an enzyme with functional
redundancy?
TLP was not the first protein to be identified as having
5-HIU hydrolytic activity. Having hypothesized the
need for additional enzymes to contribute to the oxida-
The evolution of the transthyretin-like protein S. C. Hennebry
5376 FEBS Journal 276 (2009) 5367–5379 ª 2009 The Authors Journal compilation ª 2009 FEBS
tion of uric acid [26], a 5-HIU hydrolase from soybean
(Glycine max) root nodules was purified [50,51]. This
5-HIU hydrolase showed greatest homology to
b-glucosidases (3.2.1.21) (members of the family of
retaining glycosidases) and has quite a different cata-
lytic mechanism to TLP in order to hydrolyse its sub-
strate. The fact that two structurally distinct proteins
have been identified as sharing the same function is
not uncommon in nature [52]. Legumes, such as
soybean, require sophisticated machinery for nitrogen
fixation. Therefore, it is perhaps not surprising that
they have evolved to possess two structurally unrelated
proteins involved in ureide synthesis. The question
remains as to whether this functional redundancy
exists in other plants, or indeed other bacteria, fungi
and metazoan organisms.
Conclusion
The evolution of TLP and transthyretin represents an
intriguing example of divergent evolution. The conser-
vation of catalytic residues at the TLP dimer–dimer
interface, demonstrated to be essential for enzymatic
activity, indicates that it is likely that all TLPs share
5-HIU hydrolytic activity. Following duplication of the

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