Many fructosamine 3-kinase homologues in bacteria are
ribulosamine⁄erythrulosamine 3-kinases potentially
involved in protein deglycation
Rita Gemayel, Juliette Fortpied, Rim Rzem, Didier Vertommen, Maria Veiga-da-Cunha and
Emile Van Schaftingen
Universite
´Catholique de Louvain, de Duve Institute, Brussels, Belgium
Fructosamine 3-kinase (FN3K) is a recently identified
enzyme that phosphorylates the Amadori products
fructosamines, leading to their destabilization and
removal from proteins [1–3]. FN3K is therefore respon-
sible for a new protein-repair mechanism. A related
mammalian enzyme (FN3K-related protein; FN3K-RP)
sharing 65% sequence identity with FN3K does not
phosphorylate fructosamines, but does phosphorylate
other ketoamines, mainly ribulosamines and erythrulos-
amines [4–6], as does the plant homologue of FN3K [6].
Fructosamines arise through a spontaneous reaction
of glucose with amines and their formation in vivo is
Keywords
deglycation; erythrose 4-phosphate;
fructosamine; glycation; ribose 5-phosphate
Correspondence
E. Van Schaftingen, UCL 7539, Avenue
Hippocrate 75, B-1200 Brussels, Belgium
Fax: +32 27 647598
Tel: +32 27 647564
E-mail: vanschaftingen@bchm.ucl.ac.be
(Received 11 April 2007, revised 15 June
2007, accepted 18 June 2007)
doi:10.1111/j.1742-4658.2007.05948.x
The purpose of this work was to identify the function of bacterial homo-
logues of fructosamine 3-kinase (FN3K), a mammalian enzyme responsible
for the removal of fructosamines from proteins. FN3K homologues were
identified in 200 (i.e. 27%) of the sequenced bacterial genomes. In 11
of these genomes, from phylogenetically distant bacteria, the FN3K homo-
logue was immediately preceded by a low-molecular-weight protein-tyro-
sine-phosphatase (LMW-PTP) homologue, which is therefore probably
functionally related to the FN3K homologue. Five bacterial FN3K homo-
logues (from Escherichia coli,Enterococcus faecium,Lactobacillus planta-
rum,Staphylococcus aureus and Thermus thermophilus) were overexpressed
in E. coli, purified and their kinetic properties investigated. Four were ribu-
losamine ⁄erythrulosamine 3-kinases acting best on free lysine and cadaver-
ine derivatives, but not on ribulosamines bound to the alpha amino group
of amino acids. They also phosphorylated protein-bound ribulosamines or
erythrulosamines, but not protein-bound fructosamines, therefore having
properties similar to those of mammalian FN3K-related protein. The
E. coli FN3K homologue (YniA) was inactive on all tested substrates. The
LMW-PTP of T. thermophilus, which forms an operon with an FN3K
homologue, and an LMW-PTP of S. aureus (PtpA) were overexpressed in
E. coli, purified and shown to dephosphorylate not only protein tyrosine
phosphates, but protein ribulosamine 5-phosphates as well as free ribulose-
lysine 5-phosphate and erythruloselysine 4-phosphate. These LMW-PTPs
were devoid of ribulosamine 3-phosphatase activity. It is concluded that
most bacterial FN3K homologues are ribulosamine ⁄erythrulosamine 3-kin-
ases. They may serve, in conjunction with a phosphatase, to deglycate
products of glycation formed from ribose 5-phosphate or erythrose 4-phos-
phate.
Abbreviations
DEAE, diethylaminoethyl; FN3K, fructosamine 3-kinase; FN3K-RP, FN3K-related protein; LMW-PTP, low-molecular-weight protein-tyrosine-
phosphatase; SP, sulfopropyl.
4360 FEBS Journal 274 (2007) 4360–4374 ª2007 The Authors Journal compilation ª2007 FEBS
well documented. By contrast, the presence of
ribulosamines in cells has not been demonstrated. We
have previously speculated that they may form through
a reaction of amines with ribose 5-phosphate, a potent
glycating agent. The resulting ribulosamine 5-phos-
phates, however, are not substrates for FN3K-RP and
they therefore need to be dephosphorylated by a
phosphatase to become a substrate of FN3K-RP
(Scheme 1). We recently purified a ribulosamine
5-phosphatase from human erythrocytes, a cell type in
which FN3K-RP is very active, and we identified this
enzyme as low-molecular-weight protein-tyrosine-phos-
phatase A (LMW-PTP-A) [7].
As homologues of FN3K are also found in bacteria
[1], where genes encoding functionally related proteins
are often arranged in operons, we proceeded to ana-
lyze bacterial genomes. In several instances, we found
that an FN3K homologue was associated in an operon
with a putative LMW-PTP. These findings led us to
express and characterize five bacterial FN3K homo-
logues and three LMW-PTP homologues, and to study
their substrate specificity.
Results
Search of FN3K homologues in databases
To identify the bacterial genomes comprising an FN3K
homologue, we performed tBLASTn searches in the
microbial genome database available at http://
www.ncbi.nlm.nih.gov. As of February 2007, 27%
(210 ⁄760) of all available genomes, and the same pro-
portion (124 ⁄453) of completely sequenced genomes,
contained an FN3K homologue. No more than one
homologue was identified per bacterial genome.
Remarkably, an FN3K homologue is present in all
Cyanobacteria, but only in some members of other
bacterial families (supplementary Table S1). For
instance, among Pasteurellaceae, Haemophilus somnus
and Actinobacillus succinogenes comprise an FN3K
homologue, but this is not the case for Haemophilus
influenzae and Actinobacillus pleuropneumoniae.AnFN3K
homologue was found in only 1 of the 38 sequenced
archaeal genomes, that of Haloarcula marismortui.
FN3K homologues were also identified in eukary-
otes. As previously described, two different homo-
logues, one closer to human FN3K and the other
closer to FN3K-RP, are present in mammals and
birds, whereas only one homologue is observed in fish
(and is closer to FN3K-RP). One single FN3K homo-
logue is present in Caenorhabditis elegans,Caenorhabd-
itis briggsae and Ciona intestinalis, and at least three
different homologues are present in Strongylocentrotus
purpuratus, but there are none in insects. Homologues
are also found in several fungi (e.g. Aspergilli, Neuro-
spora crassa,Magnaporthe grisea) although not in the
yeasts Saccharomyces cerevisiae and Schizosaccharomy-
ces pombe. Among protozoa, a homologue is found in
Giardia lamblia and Trypanosoma cruzi, but none in
two other trypanosomatids, Trypanosoma brucei and
Leishmania major.
The sequences were aligned by ClustalX and a
neighbour-joining tree was constructed (Fig. 1). Bacte-
rial sequences formed several clusters corresponding
mostly to known groups of bacteria [e.g. Actinobacte-
ria, Cyanobacteria (two clusters) and bacteria of the
gamma subdivision (Enterobacteriales, Pasteurellaceae,
Vibrionaceae)]. Eukaryotic sequences formed one sin-
gle cluster, with the exception of the FN3K homo-
logue of T. cruzi, which clustered with bacterial
sequences.
Genome context
We also examined the genome context of the bacterial
FN3K homologues, as this could point to functionally
CH
2
O
C
HCOH
CH
2
OH
HCOH
NH
Ribulosamine
HC
HCOH
HCOH
CH
2
HCOH
OO
-
P
O
-
O
O
NH
2
Ribose-5-P
Protein
CH
2
O
C
HCOH
CH
2
HCOH
NH
2
OO
-
P
O
-
O
Ribulosamine-5-P
CH
OC
HCOH
CH
2
OH
HC
NH
2
OO
-
P
O
-
O
Ribulosamine-3-P
HC
O
C
HCOH
CH
2
OH
HCH
O
NH
2
4,5-dihydroxy-
1,2-pentanedione
+
FN3K
homologue
ATP
ADP
LMW-PTP
Pi
H
2
O
H
2
O
Pi H
2
O
Scheme 1. Formation and repair of ribulosamines. Ribulosamines
presumably result from the reaction of amines with ribose 5-phos-
phate, followed by enzymatic dephosphorylation of ribulosamine
5-phosphates by a phosphatase. Ribulosamines are phosphorylated
by fructosamine 3-kinase (FN3K) homologues, which leads to their
destabilization and recovery of the unmodified amine. Erythrulosam-
ines presumably form in a similar manner from erythrose 4-phos-
phate (data not shown).
R. Gemayel et al. Bacterial fructosamine 3-kinase homologues
FEBS Journal 274 (2007) 4360–4374 ª2007 The Authors Journal compilation ª2007 FEBS 4361
related proteins and therefore provide information on
the origin of the substrate(s) or on the fate of the
product(s) of the FN3K homologues. Except for evolu-
tionarily related bacteria, this genome context is extre-
mely variable. However, the gene encoding the FN3K
homologue is immediately preceded by a putative
LMW-PTP in 11 genomes from phylogenetically dis-
tant bacteria: Cytophaga hutchinsonii,Thermus thermo-
philus (Fig. 2), Acidothermus cellulolyticus,Fulvimarina
pelagi,Gloeobacter violaceus,Microscilla marina,
0.1
Yersinia pestis
Photorhabdus luminescens
Erwinia carotovora
Escherichia coli
Salmonella enterica
Pasteurella multocida
Haemophilus somnus
Mannheimia succiniciproducens
Vibrio parahaemolyticus
Vibrio vulnificus
Vibrio cholerae
Vibrio fischeri
Photobacterium profundum
Pseudoalteromonas haloplanktis
Colwellia psychrerythraea
Anabaena variabilis
Nostoc punctiforme
Thermosynechococcus elongatus
Crocosphaera watsonii
Synechocystis sp.
Trichodesmium erythraeum
Gloeobacter violaceus
Synechococcus elongatus
Nitrosomonas europaea
Azoarcus sp.
Thiobacillus denitrificans
Thiomicrospira crunogena
Synechococcus sp.
Prochlorococcus marinus str.
Prochlorococcus marinus
Prochlorococcus marinus subs.
Microbulbifer degradans
Staphylococcus aureus
Staphylococcus epidermidis
Lactobacillus casei
Oenococcus oeni
Lactobacillus plantarum
Leuconostoc mesenteroides
Trypanosoma cruzi
Enterococcus faecium
Cytophaga hutchinsonii
Salinibacter ruber
Gallus gallus FN3K-RP
Homo sapiens FN3K-RP
Danio rerio
Homo sapiens FN3K
Gallus gallus FN3K
Strongylocentrotus purpuratus
Caenorhabditis briggsae
Aspergillus fumigatus
Neurospora crassa
Arabidopsis thaliana
Giardia lamblia
Thermobifida fusca
Nocardia farcinica
Corynebacterium efficiens
Corynebacterium glutamicum
Mycobacterium avium
Propionibacterium acnes
Nocardioides sp.
Bifidobacterium breve
Bifidobacterium longum
Thermus thermophilus
Chromohalobacter salexigens
Zymomonas mobilis
Rhodobacterales bacterium
Rubrobacter xylanophilus
Rhodospirillum rubrum
Haloarcula marismortui
+
+
*
**
*
*
+
*
*
+
+
*
*
*
*
*
*
+
+
Associated
LMW-PTP
(distance in bp)
yes (-19)
yes (0)
yes (17)
yes (12)
yes (-3)
yes (-37)
yes (66)
yes (-10)
Associated
YniC
(distance in bp)
yes (728)
yes (724)
yes (918)
yes (785)
yes (1094)
yes (335)
yes (0)
Cyanobacteria
Enterobacteriales
Pasteurellaceae
Vibrionaceae
Cyanobacteria
Lactobacillales
Eukaryote
Eukaryotes
Actinobacteria
Archaea
yes
Ribulosamine
3-kinase
activity
no
yes
yes
yes
yes
yes
yes
yes
yes
yes
Fig. 1. Fructosamine 3-kinase (FN3K) homologues: neighbour-joining tree, activity and association with putative phosphatases in various bac-
terial genomes. The Haloarcula marismortui sequence was used as an outgroup. Symbols at the nodes represent the support for each node
as obtained by 1000 bootstrap samplings: (*), > 95%; (+), 80–95%; (·), 50–80%. Nodes with no symbol were found in < 50% of the boot-
strap samplings. The branch lengths are proportional to the number of substitutions per site. The horizontal bar represents 0.1 substitutions
per site. The first column indicates the proteins that have been shown to phosphorylate ribulosamines in this work (framed) or in previous
work. The last two columns indicate the presence of homologues of low-molecular-weight protein-tyrosine-phosphatase (LMW-PTP) or the
phosphatase YniC close to the FN3K homologue in bacterial genomes. The figure between parentheses indicates the distance (in base pairs)
separating the two ORFs. Negative values mean that the two sequences partially overlap.
Bacterial fructosamine 3-kinase homologues R. Gemayel et al.
4362 FEBS Journal 274 (2007) 4360–4374 ª2007 The Authors Journal compilation ª2007 FEBS
Nocardioides sp., Rubrobacter xylanophilus,Salinibacter
ruber,Thermobifida fusca and Zymomonas mobilis (the
second column of Fig. 1, and data not shown). The
short distance between the two ORFs (average distance
15 nucleotides) and their identical orientation suggest
that they belong to the same operon. In another gen-
ome (from Rhodospirillum rubrum), the sequences
encoding the LMW-PTP and FN3K homologues are
separated by an ORF of 550 bp on the other strand
(data not shown).
blast searches with the Escherichia coli protein-
tyrosine kinase wzc [8] did not indicate the presence
of a homologue of this enzyme in several bacteria
containing the putative LMW-PTP ⁄FN3K operon
(A. cellulolyticus, Nocardioides sp., S. ruber,T. fusca,
T. thermophilus and Z. mobilis). This makes the
presence of an LMW-PTP homologue all the more
intriguing.
Another potentially interesting association observed
in other genomes is that of the FN3K homologue
with a phosphatase (YniC) belonging to the HAD
family and shown to act, in E. coli, on a variety of
phosphate esters [9]. The FN3K homologue is imme-
diately followed by this phosphatase in the genomes
of Photobacterium profundum and Mannheimia succi-
niciproducens and is separated from it by an ORF in
the other orientation (YniB, called YfeE in Yersinia
pestis, or homologues) in E. coli (Fig. 2), Erwinia
carotovora,Salmonella enterica and various Shigella
and Yersinia species (data not shown). The phospha-
tase YniC is, however, absent from the genomes of
most Vibrionaceae (which comprise an FN3K homo-
logue) (Fig. 1), but present in other bacteria of the
gamma subdivision (various Shewanella species,
Marinomonas sp.) that do not comprise an FN3K
homologue. It is therefore likely that the phosphatase
YniC, contrary to LMW-PTP, is not functionally
related to FN3K homologues.
Sequence alignments
Figure 3 shows an alignment of the five bacterial pro-
teins that have been biochemically characterized in the
present work with those of eukaryotic FN3K or
FN3K-RP that have been previously studied (human
FN3K and FN3K-RP; the FN3K homologue of Ara-
bidopsis thaliana) [1,4,6,10]. All sequences share several
conserved motifs. The most striking one is the nucleo-
tide-binding motif (LHGDLWxGN; residues 214–222
in the human FN3K sequence), which is similar to that
found in aminoglycoside kinases (LHxDLHxxN). Ver-
tebrate FN3Ks and FN3K-RPs contain a stretch of
about 20 residues (residues 118–140 in human FN3K)
that is absent from the prokaryotic sequences and
from the eukaryotic sequences of plants, fungi and
protists. In relation with the lack of activity of the
E. coli FN3K homologue (see below), it is interesting
to point out that its sequence differs from the others
at several positions that are conserved in all other
sequences: Ser131 (replacing Gly); Arg142 (replacing
Asp or Glu); Gln231 (replacing Phe); Arg264 (replac-
ing His); and His272 (replacing Tyr).
Action of bacterial FN3K homologues on LMW
substrates
Five bacterial FN3K homologues, from Enterococcus
faecium,E. coli,Lactobacillus plantarum,Staphylococ-
cus aureus, and T. thermophilus, which share about
30% sequence identity with the human enzyme and
30–40% sequence identity among them, were expressed
in E. coli. They were purified to homogeneity and their
kinetic properties were investigated. All bacterial
FN3K homologues, except for that from E. coli, phos-
phorylated LMW ribulosamines and erythrulosamines
(Table 1), but not fructosamines (data not shown).
Ribulosamines and erythrulosamines bound to the
Escherichia
coli
FN3K
PFKb
Outer
Membrane
Protein
Hypothetical
protein Hydrolase
YniCYniB
Thermus
thermophilus
FN3KLMW-PTP
Histidine
kinase
IndA
protein
Hypothetical
protein
GTP
binding
protein
Cytophaga
hutchinsonii
FN3KLMW-PTP
Fe uptake
regulator
Alkyl
hydroperoxide
reductase
Hypothetical
proteins
Fig. 2. Genomic environment of some bac-
terial fructosamine 3-kinase (FN3K) homo-
logues.The genomic arrangements are
shown for the FN3K homologues of Ther-
mus thermophilus,Cytophaga hutchinsonii
and Escherichia coli. The most significant
finding was the association of the FN3K
homologue with a low-molecular-weight
protein-tyrosine-phosphatase (LMW-PTP)
homologue.
R. Gemayel et al. Bacterial fructosamine 3-kinase homologues
FEBS Journal 274 (2007) 4360–4374 ª2007 The Authors Journal compilation ª2007 FEBS 4363
epsilon-amino group of lysine or to cadaverine
(decarboxylated lysine) were substrates for these
enzymes, whereas the ribulosamines bound to the
alpha-amino groups of glycine, leucine and valine were
not (data not shown). Erythrulosamines were better sub-
strates than ribulosamines as indicated by the 6–20-fold
higher catalytic efficiencies observed with erythrulose-
lysine than with ribuloselysine. d-ribulose, d-erythru-
lose and reduced ribuloselysine (pentitollysine), all
tested at 1 mm, were not phosphorylated by the
L. plantarum FN3K homologue.
To check the position of the phosphorylated carbon,
ribuloselysine was phosphorylated by the S. aureus
FN3K homologue, and the phosphorylation product
was purified and analysed by tandem mass spectrome-
try, as previously described [6]. The same fragmenta-
tion spectrum was observed [6]. In particular,
fragments of m⁄z349 and 319 were found, which indi-
cated that the third carbon of the sugar moiety was
phosphorylated.
The E. coli FN3K homologue was inactive on all
the above-mentioned compounds, including ribulose-
lysine and erythruloselysine. It was also inactive on
more than 50 other potential phosphate acceptors,
including d-ribulose, d-xylulose, choline, ethanol-
amine, l-serine, hydroxypyruvate, d-glycerate, thia-
mine and dl-homoserine (tested at concentrations of
0.1–5 mm).
Fig. 3. Alignment of human fructosamine
3-kinase (FN3K) and fructosamine 3-kinase-
related protein (FN3K-RP) with the bacterial
homologues investigated in the present
study. The sequences were aligned using
CLUSTALX. Conserved residues are high-
lighted and the residues that differ in the
Escherichia coli FN3K homologue sequence
are underlined. The abbreviations used are:
FN3K (human FN3K), FN3KRP (human
FN3K-RP), ARATH (FN3K homologue from
Arabidopsis thaliana), ECOLI (Escherichia
coli), ENTFAE (Enterococcus faecium),
LACTPL (Lactobacillus plantarum), STAPH
(Staphylococcus aureus) and THERM (Ther-
mus thermophilus).
Bacterial fructosamine 3-kinase homologues R. Gemayel et al.
4364 FEBS Journal 274 (2007) 4360–4374 ª2007 The Authors Journal compilation ª2007 FEBS