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 rib
Trang 1ribulosamine ⁄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 ribulosamcadaver-ines 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 coliFN3K 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 erythruloseribulose-lysine 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.
Trang 2well 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 homohomo-logues, 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 influenzaeand Actinobacillus pleuropneumoniae An FN3K 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
CH2 O C
HCOH
CH2OH HCOH NH
Ribulosamine
HC
HCOH
HCOH
CH2
HCOH
O P O
-O
-O
O
NH2
Ribose-5-P
Protein
CH2 O C
HCOH
CH2 HCOH
NH2
O P O
-O -O
Ribulosamine-5-P
CH O C
HCOH
CH2OH HC
NH2
O P O
-O -O
Ribulosamine-3-P
HC
O
C
HCOH
CH2OH
HCH
O
NH2
4,5-dihydroxy-1,2-pentanedione
+
FN3K homologue
ATP ADP
LMW-PTP
Pi
H2O
H2O
Pi H2O
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).
Trang 3related 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 (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
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.
Trang 4Nocardioidessp., 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 (replac(replac-ing 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 YniB YniC Hydrolase
Thermus thermophilus
FN3K LMW-PTP
Histidine kinase
IndA protein
Hypothetical protein
GTP binding protein
Cytophaga hutchinsonii
FN3K LMW-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.
Trang 5epsilon-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 ribuloseerythrulose-lysine d-ribulose,
d-erythru-lose and reduced ribud-erythru-loselysine (pentitollysine), all
tested at 1 mm, were not phosphorylated by the
L plantarumFN3K 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⁄ z 349 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 erythruloseribulose-lysine 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
C LUSTAL X 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).
Trang 6Action of bacterial FN3K homologues on
protein-bound ketoamines
We also tested the ability of the bacterial FN3K
homologues to phosphorylate protein-bound
ribulosam-ines Two proteins, hen egg lysozyme and E coli
thio-redoxin A, were glycated with ribose and used as
substrates (Fig 4) All four active bacterial FN3K
homologues and mouse FN3K catalysed the
phosphor-ylation of protein-bound ribulosamines, although their
relative activity was dependent on the substrate used
With glycated lysozyme, the most active enzyme was
the S aureus FN3K homologue, and the least active
enzyme was the one from T thermophilus Glycated
thioredoxin A was best phosphorylated by mouse
FN3K, which apparently had access to more glycation
sites than its bacterial homologues
The initial rate of the reaction with lysozyme-bound
ribulosamines was a hyperbolic function of the
sub-strate concentration in the case of the S aureus
enzyme, for which Kmand Vmaxvalues could therefore
be determined (Table 1) The other enzymes were not
saturated at the highest concentration of
lysozyme-bound ribulosamines that we tested (500 lm) and their
activities at a substrate concentration of 100 lm are
presented in Table 1 Protein-bound ribulosamines
were poorer substrates than free ribuloselysine: from
the kinetic data, it can be calculated that the activity
on lysozyme-bound ribulosamines amounted to 10%
of the activity observed with free ribuloselysine at the
same concentration
Lysozyme-bound erythrulosamines were also
phos-phorylated by the four active bacterial FN3K
homo-logues at rates that were about twofold higher
(E faecium), or two- to fourfold lower (all others), than those observed with lysozyme-bound ribulosamines Lysozyme-bound fructosamines were not detectably Table 1 Kinetic properties of the bacterial fructosamine 3-kinase (FN3K) homologues The results are the means of two or three determina-tions In the latter case, the SEM value is given V max values are expressed as nmol phosphorylated product formed per min and per mg of protein E faecium, Enterococcus faecium; L plantarum, Lactobacillus plantarum; ND, not determined; S aureus, Staphylococcus aureus;
T thermophilus, Thermus thermophilus.
Substrate
Km (l M )
Vmax (nmolÆmin)1Æmg)1)
Km (l M )
Vmax (nmolÆmin)1Æmg)1)
Km (l M )
Vmax (nmolÆmin)1Æmg)1)
Km (l M )
Vmax (nmolÆmin)1Æmg)1)
Ribulosamine-lysozyme
> 500 16 ± 0.3 a > 500 40 ± 4 a 44 ± 6 220 ± 25 > 500 7 ± 1 a
Ribulosamine-Thioredoxin A
Erythrulosamine-lysozyme
> 500 3.8 ± 0.1 a > 500 80 ± 6 a 42 ± 4 78 ± 2 > 500 4.2 ± 0.2 a
a Activity at 100 l M protein-bound ribulosamine or erythrulosamine.
0.00 0.05 0.10 0.15
0.20
A
B
FN3K
S aureus
E faecium
M musculus
T thermophilus
-L plantarum
Time (min)
Time (min)
Incorporated Phosphate (mol P/mol ribulosamines)
Incorporated Phosphate (mol P/mol ribulosamines)
0.00 0.05 0.10
S aureus
E faecium
M musculus
T thermophilus
-L plantarum
Fig 4 Phosphorylation of protein-bound ribulosamines by mouse fructosamine 3-kinase (FN3K) and four bacterial FN3K homologues Lysozyme (A) and Escherichia coli thioredoxin A (B) glycated with ribose were used as substrates at 50 l M protein-bound ribulosa-mines The samples were incubated with [ 32 P]ATP[cP] and
50 lgÆmL)1of each FN3K homologue Incorporated phosphate was measured at different time-points The results are the means of three independent measurements ± SEM.
Trang 7phosphorylated by the enzymes from E faecium and
L plantarum, but they were slowly phosphorylated by
the enzymes from S aureus and T thermophilus, at
rates corresponding to 0.4 and 2%, respectively, of the
activity observed with lysozyme-bound ribulosamines
None of these enzymes catalysed the phosphorylation
of protein-bound ribulosamine 5-phosphates (data not
shown) The E coli FN3K homologue was also
inac-tive on all macromolecular substrates tested, which
included lysozyme-bound d- and l-ribulosamines,
d-ribulosamine 5-phosphates, fructosamines and
erythrulosamines (data not shown)
The product of the phosphorylation of
lysozyme-bound ribulosamines by the FN3K homologues from
S aureusand T thermophilus broke down with a
half-life of 26–28 min at 37C and neutral pH (data not
shown), as previously observed with the product of
human FN3K-RP [5] and the plant FN3K homologue
[6] These results further indicated that bacterial FN3K
homologues also phosphorylated carbon 3 of the sugar
moiety of ribulosamines
Substrate specificity of the LMW-PTP
homologues
We expressed the LMW-PTP homologue belonging to
the same operon as the FN3K homologue in the T
ther-mophilusgenome, as well as the two LMW-PTP
homo-logues, PtpA and PtpB [11], present in the S aureus
genome PtpA and PtpB, which share, respectively, 38
and 28% sequence identity with T thermophilus
LMW-PTP, are encoded by genes that are distant from the
gene encoding the FN3K homologue and do not
appar-ently belong to operons All three recombinant proteins
were purified to homogeneity and their activities tested
both on LMW and macromolecular substrates
S aureus PtpB was poorly active or inactive on all
substrates tested, in agreement with previous results
[11] The other two enzymes dephosphorylated
previ-ously described substrates for LMW-PTP
(p-nitro-phenyl phosphate, FMN), but also ribuloselysine
5-phosphate and erythruloselysine 4-phosphate, the
T thermophilusenzyme being particularly active on the
latter substrate (Table 2) They did not act on ribose
5-phosphate, fructose 6-phosphate or glucose
6-phos-phate (data not shown) We checked that
dephosphory-lation of ribuloselysine 5-phosphate by S aureus
LMW-PTP (PtpA) led to the formation of a substrate
for a bacterial FN3K homologue (the one from E
fae-cium was used in this experiment) That the resulting
phosphorylation product was ribuloselysine
3-phos-phate was indicated by its instability and by the
fact that its decomposition led to the appearance of
4,5-dihydroxy-1,2-pentanedione, as determined by mass spectrometry analysis of the quinoxaline derivative [6] The activity of LMW-PTPs on protein substrates was tested through the release of32P from radiolabelled sub-strates As shown in Fig 5, T thermophilus LMW-PTP acted about 10-fold faster on protein tyrosine-phos-phates than on protein ribulosamine 5-phostyrosine-phos-phates, whereas S aureus PtpA acted preferentially on the latter substrate S aureus PtpB was also poorly active on pro-tein substrates As illustrated for T thermophilus LMW-PTP, dephosphorylation of lysozyme glycated with ribose 5-phosphate by this phosphatase led to the for-mation of a substrate for the S aureus FN3K homo-logue (Fig 6) The resulting phosphorylation product was unstable and broke down, at 37C, with a half-life similar to that of ribulosamine 3-phosphates (data not shown) Similarly, incubation of lysozyme-bound erythrulosamine 4-phosphates with T thermophilus LMW-PTP or S aureus PtpA led to the formation of a substrate for the S aureus FN3K homologue (Fig 7)
Discussion Most bacterial FN3K homologues are ribulosamine⁄ erythrulosamine 3-kinases Four of the five bacterial FN3K homologues that we studied are ribulosamine⁄ erythrulosamine 3-kinases This property is shared by mammalian and avian FN3Ks and FN3K-RPs, as well as by the single FN3K homologue present in fish and plants This observation leads us to the conclusion that the ances-tral ‘FN3K’ protein was probably a ribulosamine⁄ ery-thrulosamine 3-kinase The ability to phosphorylate
Table 2 Activities of low-molecular-weight protein-tyrosine-phos-phatase (LMW-PTP) homologues on LMW substrates Substrates were assayed at 0.5 m M final concentration Activities are expressed as nmol inorganic phosphate formed per min and per
mg of protein The data represent the means of three values ± SEM ND, not detectable; Ptp, LMW-PTP of S aureus; S aureus, Staphylococcus aureus; T thermophilus, Thermus thermophilus.
Substrate
Enzyme activity (nmolÆmin)1Æmg of protein)
T thermophilus
S aureus (PtpA)
S aureus (PtpB) p-Nitrophenyl phosphate 1820 ± 140 7750 ± 230 42 ± 1 Flavin mononucleotide 1730 ± 130 16900 ± 520 24 ± 3
Ribuloselysine-5-phosphate
Erythruloselysine-4-phosphate
5300 ± 630 1470 ± 100 ND
Trang 8fructosamines, which is restricted to mammalian and
avian FN3Ks, was acquired late in evolution following
a gene duplication event that took place in the lineage
leading to mammals and birds [10] It is not known at
present if the E coli homologue is an inactive protein
or if it has acquired a distinct substrate specificity
The physiological substrate of the active bacterial
FN3K homologues is presently not known, but its
structure is presumably close to that of a ribulosamine
or an erythrulosamine The observations that no
phos-phorylation is observed with ribulose, with the reduced
forms of ribuloselysine and with xyluloselysine (C3
epi-mer of ribuloselysine), stress the importance of the
presence of an amino group on C1, a keto function on
C2 and a hydroxyl group with a D configuration on
C3 In addition, as initially observed with FN3K [12],
ketoamine derivatives bound to the alpha amino group
of amino acids are poor substrates, whereas
ketoam-ines bound to the epsilon amino group of lysine or
cadaverine are excellent substrates
Bacterial FN3K homologues are more than 10-fold
more active on LMW ketoamines than on
protein-bound ketoamines, which suggests that their
physiolog-ical substrates are LMW compounds However, their
absolute activity on protein substrates is higher than
that of mammalian FN3K or FN3K-RP on similar substrates For instance, the Vmax of fructosamine 3-kinase when it acts on lysozyme-bound fructosam-ines amounts to 10 nmolÆmin)1Æmg)1 of protein (G Delpierre, E Van Schaftingen, unpublished results), which is about 20-fold lower than the Vmax of the
S aureus enzyme for protein-bound ribulosamines As FN3K and FN3K-RP have been shown to be involved
in protein repair in vivo or in intact cells [2,3,5], this comparison suggests that this may also be true for their bacterial homologues
Endogenous or exogenous source for the substrates of bacterial FN3K homologues?
The specificity of the FN3K homologues indicates that they act on sugar derivatives The latter could either
be of internal or external origin The absence of associ-ation of FN3K homologues with a transporter does not support the idea that they play a role in the metabolism of an exogenous substrate This is unlike fructosamine-6-phosphate deglycases, which are almost always encoded by operons also containing genes for putative fructosamine transporters [13,14] It is also conceivable that the substrate for the FN3K
0 10 20 30 40 50 60 70 80 90 100
A
B
Enzyme Substrate
Enzyme Substrate
Lysozyme-RN5P MBP-TyrP
50 µg.mL -1
5 µg.mL -1
Lysozyme-RN5P MBP-TyrP
-Time (min)
0 10 20 30 40 50 60 70 80
PtpA 10 µg.mL -1
PtpA 10 µg.mL -1
PtpB 50 µg.mL-1 PtpB 50 µg.mL -1
Lysozyme-RN5P
MBP-TyrP
Lysozyme-RN5P MBP-TyrP
Time (min)
Fig 5 Dephosphorylation of protein
tyro-sine-phosphates and protein ribulosamine
5-phosphates by bacterial LMW-PTP
homo-logues Thermus thermophilus (A) and
Staphylococcus aureus (B)
low-molecular-weight protein-tyrosine-phosphatase
(LMW-PTP) homologues were used to
dephosphorylate myelin basic protein-bound
[32P]tyrosine phosphates (MBP-TyrP) and
lysozyme-bound [ 32 P]ribulosamine
5-phos-phates (Lysozyme-RN5P), both tested at
2 l M protein-bound [32P]phosphate The
concentration of each homologue used is
shown on the graph, and conditions where
no LMW-PTP was added are shown in open
symbols The radioactivity, corresponding to
32 P inorganic phosphate, released after
trichloroacetic acid precipitation of proteins
was measured at different time-points The
results are the means of three independent
measurements ± SEM.
Trang 90 20 40 60 80 100
0.0
0.1
0.2
0.3
0.4
0.5
A
T thermophilus
LMW-PTP Ribose-5-P
LMW-PTP Ribose-5-P
20 m M
-0 m M
20 m M
Time (min)
0.00
0.01
0.02
0.03
0.04
0.05
B
T thermophilus
T thermophilus
20 m M
-T thermophilus 0 m M
20 m M
Time(min)
Incorporated Phosphate (mol P/mol lysozyme)
Fig 6 Dephosphorylation of protein ribulosamine 5-phosphates by the Thermus thermophilus low-molecular-weight protein-tyrosine-phosphatase (LMW-PTP) homo-logue and rephosphorylation by a bacterial fructosamine 3-kinase (FN3K) homologue (A) Lysozyme (5 mgÆmL)1) glycated with
20 m M ribose 5-phosphate was incubated with 60 lgÆmL)1of T thermophilus LMW-PTP (closed circles), or without LMW-LMW-PTP (open circles) Unglycated lysozyme was also incubated with T thermophilus LMW-PTP (closed diamonds) The liberated inor-ganic phosphate was measured at different time-points (B) The resulting product of dephosphorylation was then incubated with [ 32 P]ATP[cP] and 50 lgÆmL)1of Staphylo-coccus aureus FN3K homologue for 15 min and the incorporated phosphate was mea-sured The results are the means of three independent measurements ± SEM.
0.0
0.1
0.2
0.3
0.4
0.5
A
B
LMW-PTP Erythrose-4-P
10 m M
S aureus PtpA
S aureus PtpA
T thermophilus
5 m M
10 m M
5 m M
T thermophilus
LMW-PTP Erythrose-4-P
S aureus PtpA
S aureus PtpA
T thermophilus
T thermophilus
10 m M
-Time(min)
Time(min)
0.00
0.02
0.04
0.06
0.08
0.10
0.12
10 m M
5 m M
10 m M
5 m M
-5 m M
10 m M Incorporated Phosphate (mol P/mol lysozyme)
Fig 7 Dephosphorylation of protein erythrulosamine 4-phosphates by bacterial low-molecular-weight protein-tyrosine-phosphatase (LMW-PTP) homologues and rephosphorylation by a bacterial fructos-amine 3-kinase (FN3K) homologue (A) Lysozyme (5 mgÆmL)1) glycated with 5
or 10 mm erythrose 4-phosphate was incubated with 20 lgÆmL)1of Thermus thermophilus LMW-PTP (open squares and triangles) or Staphylococcus aureus protein-tyrosine-phosphatase A (closed squares and triangles) or without LMW-PTP (open circles and diamonds) Unglycated lysozyme was also incubated with T thermophilus LMW-PTP (closed circles) The liberated inorganic phosphate was measured at different time-points (B) The resulting product of dephosphorylation was then incubated with [32P]ATP[cP] and 50 lgÆmL)1of S aureus FN3K for 5 min and the incorporated phosphate was measured The results are the means of three independent measurements ± SEM.
Trang 10homologues is an exogenous, toxic compound, which,
like aminoglycosides and macrolides, would have to be
inactivated by phosphorylation [15] To the best of our
knowledge, no known antibiotic is a ketoamine
deriva-tive, but hypothetical ketoamine antibiotics may have
been missed in the screenings for antibacterial
com-pounds because of their instability
An endogenous origin for the substrate(s) of FN3K
homologues has therefore to be considered However,
except for the association with an LMW-PTP (see
below), the context in which FN3K homologues are
found in bacterial genomes is not suggestive of any
pathway leading to the formation of a ketoamine
The association with a phosphatase suggests
that ribulosamines are formed from ribulosamine
5-phosphates
We have previously speculated that ketoamines may
arise through glycation of amino compounds by ribose
5-phosphate or erythrose 4-phosphate, which are
potent glycating agents that occur physiologically in
all cell types The ribulosamine 5-phosphates and
ery-thrulosamine 4-phosphates that are so formed are not
substrates for mammalian or bacterial FN3K
homo-logues A phosphatase is therefore needed to remove
the terminal phosphate before these kinases can act
(Scheme 1) We recently purified a ribulosamine
5-phosphatase from human erythrocytes and identified
it as LMW-PTP-A [7]
Interestingly, an LMW-PTP homologue forms an
operon with the FN3K homologue in 11 genomes
from phylogenetically distant bacteria This association
was not the result of recent lateral transfer events, as
indicated by the fact that the homologues of FN3K
and LMW-PTP present in these operons are very
dis-tant proteins This operon is therefore either an
extre-mely ancient operon that has been conserved or the
result of distinct recombination events that took place
independently in several bacterial lineages Both
expla-nations argue strongly for the physiological relevance
of this association
The LMW-PTP homologue of T thermophilus, and
one of the two homologues (PtpA) of S aureus,
dephosphorylated not only phosphotyrosine, but also
ribulosamine 5-phosphates and erythrulosamine
4-phosphates, converting them to substrates for bacterial
FN3K homologues The apparent absence of a
bacterial-type tyrosine kinase in several of the bacterial
genomes containing the putative LMW-PTP⁄ FN3K
operon suggests that the LMW-PTP homologues may
serve physiologically to dephosphorylate substrates
dif-ferent from protein-tyrosine phosphates
We considered the possibility that the FN3K homo-logues which we studied could be tyrosine kinases However, no phosphorylation was observed when these enzymes were allowed to act upon proteins that had not been glycated with ribose or erythrose (data not shown) Interestingly, LMW-PTP did not display detectable ribulosamine 3-phosphate phosphatase activity, which indicates that its function is not to antagonize the activity of the FN3K homologues, but,
on the contrary, to complement it The most probable hypothesis is therefore that the phosphatase functions physiologically as a ribulosamine 5-phosphate⁄ erythru-losamine 4-phosphate phosphatase, allowing the for-mation of substrates for the FN3K homologues
Potential sources of ribulosamine 5-phosphates and erythrulosamine 4-phosphates
Ribose 5-phosphate and erythrose 4-phosphate are potent glycating agents, reacting with proteins about 80- and 500-fold more rapidly than glucose, respec-tively [7,16; R Gemayel, unpublished results] The information on the concentration of these phosphate esters in bacteria is scant The xylulose 5-phosphate content of Oenococcus oeni (previously known as Leu-conostoc oenos) amounts to 0.1–0.33 lmolÆg)1 dry weight, corresponding to concentrations of about 0.033–0.1 mm [17] The concentration of ribose 5-phos-phate is probably of the same order of magnitude, indicating that in this bacterium, the glycating power
of ribose 5-phosphate is comparable to that of 2 mm glucose Erythrose 4-phosphate is likely to accumulate
in bacteria under some conditions, for example in the absence of O2 in O oeni This bacterium forms sub-stantial amounts of erythritol under this condition, because phosphoketolase acts then on fructose 6-phos-phate rather than on xylulose 5-phos6-phos-phate and there-fore forms erythrose 4-phosphate (and acetyl-phosphate) [17]
Although we have no proof at this stage that FN3K and LMW-PTP homologues participate in the repair
of glycation adducts made from ribose 5-phosphate and erythrose 4-phosphate, indirect arguments support this hypothesis One is the consistent presence of FN3K and LMW-PTP homologues (Table S1) in Cy-anobacteria, which are dependent on the Calvin cycle This is reminiscent of the high ribulosamine 3-kinase activity found in spinach leaves and the fact that plant FN3K homologues are targeted to chloroplasts [6] Furthermore, the glycation repair hypothesis may offer
a unitary explanation for the presence of enzymes with
a similar function in organisms or cells that are so dis-similar as bacteria, plants and erythrocytes Unlike