UMP kinase from the Gram-positive bacterium Bacillus subtilisis strongly dependent on GTP for optimal activity 1 Laboratoire de Chimie Structurale des Macromole´cules,4Unite´ de Ge´ne´ti
Trang 1UMP kinase from the Gram-positive bacterium Bacillus subtilis
is strongly dependent on GTP for optimal activity
1
Laboratoire de Chimie Structurale des Macromole´cules,4Unite´ de Ge´ne´tique des Ge´nomes Bacte´riens, Institut Pasteur, Paris, France,2Laboratory of Enzymology and Applied Microbiology, Cantacuzino Institute, Bucharest, Romania,
3
Centre de Biochimie Structurale, Faculte´ de Pharmacie, Universite´ de Montpellier I, Montpellier, France
The gene encoding Bacillus subtilis UMP kinase (pyrH/
smbA) is transcribed in vivo into a functional enzyme,
which represents approximately 0.1% of total soluble
proteins The specific activity of the purified enzyme under
absence of GTP, the activity of B subtilis enzyme is less
than 10% of its maximum activity Only dGTP and
(dGTP) can increase catalysis significantly Binding of
Ant-dGTP to B subtilis UMP kinase increased the quantum
yield of the fluorescent analogue by a factor of more than
three UTP and GTP completely displaced Ant-dGTP,
whereas GMP and UMP were ineffective UTP inhibits
UMP kinase of B subtilis with a lower affinity than that
shown towards the Escherichia coli enzyme Among nucleoside monophosphates,5-fluoro-UMP (5F-UMP)
corresponding nucleosides towards this bacterium A structural model of UMP kinase,based on the conser-vation of the fold of carbamate kinase and N-acetyl-glutamate kinase (whose crystals were recently resolved), was analysed in the light of physicochemical and kinetic differences between B subtilis and E coli enzymes Keywords: UMP kinase; B subtilis; molecular modelling; GTP activation; fluorescent markers
Phosphorylation of UMP and CMP in eukaryotes is carried
out by a single protein UMP/CMP kinases from
adeny-late kinase from muscle cytosol [1–5] Enteric bacteria
contain separate CMP and UMP kinases,and mutants of
corresponding genes (mssA/cmk and
pyrH/smbA,respect-ively) were isolated and characterized many years ago [6–8]
Recombinant CMP and UMP kinases from E coli have
been characterized in detail [9–15] The CMP kinase from
dCMP [11] Despite the little overall sequence identity with
other known nucleoside monophosphate (NMP) kinases,
CMP kinase from E coli has,in common with these
enzymes,a central parallel b-sheet,the strands of which are
connected by a-helices [13] In contrast,the UMP kinase from E coli is a homohexamer whose primary structure diverges from that of other NMP kinases,and is controlled allosterically by GTP (activator) and UTP (inhibitor) [9] Attempts,in the past,to isolate a specific UMP kinase from Bacillus subtilis were unsuccessful It was suggested that phosphorylation of UMP in this bacterium is accom-plished by a CMP kinase with a broader specificity for pyrimidine nucleotides than the enzyme from E coli [16] The deleterious effect of disruption of cmk/jofC gene in
B subtilis[17],and the kinetic properties of the correspond-ing recombinant protein,were in line with this interpre-tation Thanks to genome sequencing programs,the pyrH gene has been identified in all bacteria investigated,inclu-ding B subtilis The pyrH gene from Lactococcus lactis,
a bacterium similar to B subtilis in the metabolism of pyrimidine nucleotides,complements a temperature-sensi-tive pyrH mutation in E coli,demonstrating the ability of the encoded protein to synthesize UDP [18]
These observations reopened the question of the role played by UMP kinase in the metabolism of B subtilis, and in Gram-positive organisms in general,and prompted
us to clone the pyrH gene from B subtilis and to examine the structural and catalytic properties of the recombinant protein When compared with the E coli UMP kinase, several striking characteristics of the B subtilis enzyme were noticed Thus,in either crude extract or in purified form,the enzyme is unstable in the absence of UTP On the other hand,the activity of B subtilis UMP kinase is very low in the absence of GTP,which explains why
Correspondence to A.-M Gilles,Laboratoire de Chimie Structurale
des Macromole´cules,Institut Pasteur,28,rue du Dr Roux,
75724 Paris cedex 15,France.
Fax: +33 1 40 61 39 63,Tel.: +33 1 45 68 89 68,
E-mail: amgilles@pasteur.fr
Abbreviations:
Ant-dGTP,3¢-anthraniloyl-2¢-deoxyguanosine-5¢-triphosphate; CK-like CPSpf,carbamate kinase-like carbamoyl
phosphate synthase from Pyrococcus furiosus; CKef,carbamate
kinase from Enterococcus faecalis; 5F-UMP,5-fluoro-UMP;
NAGKec, N-acetylglutamate kinase from Escherichia coli.
(Received 10 March 2003,revised 20 May 2003,
accepted 3 June 2003)
Trang 2previous attempts to isolate the enzyme from this
organism were unsuccessful
Materials and methods
Chemicals
Nucleotides,restriction enzymes,T4 DNA ligase,T7 DNA
polymerase and coupling enzymes were from
Roche-Applied Science or from Sigma NDP kinase from D
I Lascu 5-Fluoro-UMP (5F-UMP) and 6-aza-UMP were
synthesized from the corresponding nucleosides with GTP
as phophoryl donor,creatine phosphate as regenerating
system and recombinant E coli uridine kinase and rabbit
muscle creatine kinase as catalysts The reaction medium in
6-aza-uridine and 5F-6-aza-uridine to nucleoside monophosphates was
>90%,the reaction medium was heated to precipitate
proteins After desalting,5-mg samples of 5F-UMP and
(Ant-dGTP) was prepared from dGTP and isatoic anhydride
essentially by the procedure described in Hiratsuka [19],for
the synthesis of Ant-dATP The analogue was purified by
chromatography on LiChroprep RP-18 (25–40 lm) using
Bacterial strains, plasmids, growth conditions
and DNA manipulations
General DNA manipulations were performed as described
by Sambrook [21] ORFs from the pyrH gene from
using the strain 168 [22] as the template and with the
following primers: 5¢-pyrH, 5¢-GGGGCATATGGAA
3¢-pyrH, 5¢-CCCCCTCGAGTTATTTCCCCCTCACGA
TCGTTCCGATTGATTCAC-3¢ The product was
inser-ted between the NdeI and XhoI restriction sites of plasmid
pET24a (Novagen) The resulting plasmid (pSL13) was
introduced into strain BL21(DE3)/pDIA17 [23] to
overpro-duce the UMP kinase The recombinant strain was grown in
2YT medium supplemented with antibiotics to an
absor-bance (A) of 1.0 at 600 nm,then overproduction was
triggered by isopropyl-b-D-thiogalactoside induction (1 mM
final concentration) for 3 h,and bacteria were harvested by
centrifugation
Purification of UMP kinase and activity assays
by centrifugation at 10 000 g for 30 min The supernatant
was concentrated by ultrafiltration,then applied to a
containing >95% pure UMP kinase was concentrated to
was purified by Nickel-nitriloacetic acid affinity chromato-graphy using the QIA express system [24] The UMP kinase
spectro-photometric assay (0.5-mL final volume) on an Eppendorf PCP6121 photometer [25] The reaction medium contained
GTP and 2 units each of lactate dehydrogenase,pyruvate kinase and NDP kinase The crude or pure preparation of
UMP The decrease in absorbance (A) at 334 nm was then recorded and corrected for secondary reactions,occurring in the absence of UMP One unit of UMP kinase corresponds
to 1 lmol of product formed per min The thermal stability
presence or absence of various nucleotides Results were expressed as percentage of activity as compared with unincubated controls
Analytical procedures Protein concentration was measured according to Brad-ford [26] Ion spray mass spectra were recorded on a quadrupole mass spectrometer,API-365 (Perkin-Elmer), equipped with an ion spray (nebulizer-assisted
20% acetonitrile in water and 0.1% HCOOH,was
SDS/PAGE was performed as described by Laemmli [27] The protein bands from SDS/PAGE were electroblotted into a Problot membrane filter (Applied Biosystems) and detected by Coomassie Blue staining The N-terminal amino acid sequence of the protein from the excised band was determined by a protein sequencer (Applied Biosys-tems,Inc.) Fluorescence experiments were performed on
Beckman Optima XLA ultracentrifuge using a AN60Ti rotor and a cell with a 12-mm optical length Samples
10 000 r.p.m Data were analysed by using the programs
IDEAL1 and IDEAL2,supplied by Beckman
Sequence comparison and molecular modelling Protein sequence database searches were performed using the PSI-BLAST,version 2.0.5,program [28] with default parameters Protein structure database searches were per-formed using a metaserver dedicated to sequence–structure comparison at low sequence identity level (15–20%) [29] Alignment refinement was performed manually and
optimization and long molecular dynamics [31] Models
Trang 3[34] Trace evolution is as analysed using CONSURF [35],
aiming to delimit the active site as well as the monomer–
monomer interface
Results
The pyrH gene from B subtilis was cloned by PCR into the
expression vector pET24a,and sequenced The resulting
ORF showed two differences when compared with the
published sequence [22]: one additional T at bp 170; and
one missing A at bp 185 As a consequence,the ORF of the
four amino acid residue changes,as follows:
57LeuTrpArg-Gly60 instead of TyrGlyAlaGlu in the original sequence
These differences are not trivial; Arg and Gly are two strictly
conserved residues in bacterial UMP kinases,the sequences
of which are available in the gene databank Substitution of
the equivalent Arg in E coli UMP kinase (Arg62His) has
pleiotropic effects on stability,catalysis or allosteric
regu-lation [12] and is responsible for the altered morphological
phenotype of E coli under nonpermissive conditions,such
as cold-sensitive growth or hypersensitivity to SDS [36] On
the other hand,Trp58 of the B subtilis enzyme is conserved
in Streptococcus pneumoniae, Strep mutans,
and may be a signature for Gram-positive organisms
(Fig 1) This amino acid is substituted in the UMP kinase
from Gram-negative bacteria by a Phe residue When
present in UMP kinases from Gram-negative organisms,
Trp is located in the middle of the sequence (Trp119 in
E coli) As the environment of Trp in UMP kinases from
Gram-positive and Gram-negative organisms is a priori
different,we expected to find differences in protein intrinsic
fluorescence properties,which was indeed the case
Harboured on high-copy number vectors,the B subtilis
strain KUR1244 (pyrH88ts) of E coli [37],indicating that it
was functional Complementation experiments performed
using E coli strain MC4100-42-14:40 (car::lacZpyrH42),in
which expression of the car::lacZ fusion is repressed in the
presence of wild-type UMP kinase activity,showed that in
high copy-number,the pyrH gene from B subtilis resulted
in a significant repression of b-galactosidase activity
From the specific activity of UMP kinase in crude
specific activity of the pure recombinant protein under
identical experimental conditions,we assumed a protein
abundance in B subtilis extracts of 0.1%,a figure close to
that found in E coli
Sequence comparison and molecular modelling
UMP kinase from B subtilis as a query sequence,first
showed sequence similarities with UMP kinases from other
bacteria Among 26 UMP kinases examined for sequence
homology (12 from positive bacteria,14 from
Gram-negative bacteria),33 strictly conserved amino acid residues,
i.e 14% of the whole sequence,were noticed The most
frequently represented residues were Gly,Asp and Arg
Site-directed mutagenesis or analysis of the phenotypically characterized mutants [36,37] showed that several strictly conserved amino acids were essential for thermodynamic stability,catalysis or allosteric regulation [12] Further iterations of thePSI-BLASTsearch revealed sequence similar-ity of bacterial UMP kinases with pyrroline-5-carboxylate synthase,glutamate-5-kinase,aspartokinases,N-acetylglu-tamate kinases and carbamate kinases (Fig 1) The latter appeared to be 18% identical with UMP kinases,while
region of 200 amino acids)
Several motifs appeared to be well conserved among these kinases At the N-terminus,a glycine-rich sequence motif (r///k/sGxA/: upper case stands for strictly conserved residue; /,for hydrophobic amino acid; and,x,for any amino acid) and a second glycine-rich motif (///GgGnx/r) appeared conserved These motifs comprise the predicted b-strands (b1 and b2) of UMP kinases,both of which might be involved in phosphate binding,according to the crystal structures of carbamate and N-acetylglutamate kinases In the first motif,the position marked k (Lys12 in UMP kinase from B subtilis) is occupied either by an alanine (carbamate kinases only) or by a lysine in all other kinases belonging to this superfamily The side-chain of this residue points toward the leaving phosphate group of ATP This suggested that carbamate kinases constitute a divergent subfamily with a slightly distinct catalytic mech-anism A third conserved stretch (r////aaGxgn),is present
at the end of the fourth predicted b-strand [38] In UMP kinases,it is immediately followed by the motif ffttDs, including a catalytic aspartate [9,12,38] The next motif (txvdGvftadPk) followed the predicted b5-strand (ead///) and provides a Thr and a Val to the active site of UMP kinases,as well as in aspartokinases and glutamate kinases This motif comprises the aspartates D168 and D174 (UMP kinase from E coli numbering) whose role in enzyme activity was also probed by site-directed mutagenesis [9,12,38] A motif (/k//Dxta) conserved among UMP kinases (including D201 in the UMP kinase from E coli) [31] would correspond to the N-terminus of the putative helix, a7,which is predicted to contribute residues to the active site The last conserved motif (gtx/) seems to stabilize the specific structure of the ATP-binding site,rather than directly participating in the binding itself,in the two solved crystal structures [39,40] The observed conservation of these motifs suggested that a similar mode of ATP binding
is used by the enzymes of this superfamily Despite clear homologies shared by these kinases,proper sequence alignment requires further refinement,based on sequence-structure comparison,owing to the low overall sequence identity level ( 15–20%)
Threading of UMP kinase from B subtilis,using the new meta-server [29],revealed significant fold compatibility (>95% confidence) with carbamate kinase from Ent
phosphate synthase from Pyrococcus furiosus (CK-like CPSpf) [39],as well as with N-acetylglutamate kinase from
was refined usingTITO[30] in order to gather the majority of insertions/deletions in loop regions CKef and CK-like CPSpf are more than 60 amino acids longer than the UMP kinase from B subtilis owing to the presence of a small
Trang 4subdomain of unknown function The latter module is
also absent in NAGKec The three-dimensional structure
of NAGKec and CK-like CPSpf were subsequently
used for molecular modelling,taking advantage of their
cocrystallization with ATP analogues However,sequence
conservation in the catalytic site also suggested closer
relatedness of UMP kinases and N-acetylglutamate kinases
(e.g: conservation of a buried lysine; K12 in B subtilis) In
CK-like CPSpf and NAGKec,the C-terminal region
participating in the binding of the phosphate donor shows
some structural and sequence variations [39,40] Refined sequence comparison in the vicinity of this region suggested that the ATP-binding site of UMP kinases more closely resembles that of NAGKec than CKs (Fig 2)
)0.7 kcal ()1.9 and )1.8 for the structures of NAGKec and
monomeric model +0.33/+0.29) were also satisfactory
Fig 1 Sequence alignment of 10 representative bacterial UMP kinases (bacsu, Bacillus subtilis;strpn, Streptococcus pneumoniae;staau, Staphylo-coccus aureus;entfe, EnteroStaphylo-coccus faecalis;myctu, Mycobacterium tuberculosis;neime, Neisseria meningitidis;pseae, Pseudomonas aeruginosa;ecoli, Escherichia coli;haein, Haemophilus influenzae;yerpe, Yersinia pestis) The sequence of N-acetylglutamate kinase from E coli,as well as of carbamate kinase-like carbamoyl phosphate synthase from Pyrococcus furiosus,whose structures were solved (PDB1gs5 and PDB1e19,respect-ively),are at the bottom Strictly conserved residues are indicated in the red box The secondary structure elements,as assigned after structure modelling,are indicated on the top of the sequences Motifs discussed in the text are written below the alignment The figure was drawn using
ESPRIPT [49].
Trang 5the quality at the atomic level (correctness scores of 73% for
UMP kinase from B subtilis vs 98% and 99% for the
structures of NAGKec and CK-like CPSpf,respectively)
Similar values were also obtained for various other UMP
kinases The proposed interface for dimerization is in
CON-SURF[35] The latter highlighted a second interface closer to
the active site that might correspond to the dimer–dimer
interactions The latter would encompass the region of
Trp119 and Pro141 in the UMP kinase from E coli,in
agreement with fluorescence and mutagenesis
data,suggest-ing that this region is implied in allosteric regulation [9,38]
Further experimental validation will be necessary to verify
these hypotheses
The present model gathers the sequence stretches that are
well conserved among UMP kinases on one face of each
monomer These faces are mostly composed of b-strand
C-termini and a-helice N-termini,and the connecting loops
The model identifies the C-terminal segment in bacterial
UMP kinases as the ATP-binding site,while the N-terminal
domain would contain the cosubstrate and the effector
binding sites The dimerization interface is composed of
a-helices and a b-strand from the N-terminal end The
current model suggests that Trp58 is readily accessible to the
solvent in a pocket opening on the active site,while Cys206
is buried by the very C-terminus (residues Gly239–Lys240)
Trp58 ATP would be in close contact with Ne of Lys12,a
residue strictly conserved in bacterial UMP kinases as well
as in the other members of this superfamily of catalysts,
such as carbamate kinases Substitution of this conserved
Lys residue in UMP kinase from Streptococcus pneumoniae yielded a completely inactive enzyme (L Assairi,unupub-lished results) The catalytic Asp146 identified in UMP kinase from E coli corresponds to Asp143 in the corresponding B subtilis enzyme (Fig 2) In the vicinity of the active site,the main difference between the latter enzyme and UMP kinases from the Gram-negative organisms is the insertion of one residue (Asn164) lying in the vicinity of ATP The putative role of this one-residue insertion remains unexplained in our current structure modelling
Purification and molecular properties of recombinant
UMP kinase from B subtilis,overproduced in strain BL21(DE3)/pDIA17,was purified as described in the Materials and methods,i.e a heating step followed by gel-permeation chromatography The molecular mass of
by ESI-MS,was in agreement with that calculated (26 083 Da) from the sequence Gel-permeation chromato-graphy yielded a peak consistent with an oligomeric enzyme (six subunits/oligomer) that was preceded by a shoulder Ultracentrifugation analysis by sedimentation equilibrium indicated that the dominant species corresponded to the hexameric enzyme (154 kDa),even though oligomers of a higher molecular mass (283 kDa) were also identified They correspond most probably to the association of two hexamers In parallel,an N-terminal His-tagged form was produced and purified by Nickel-nitriloacetic chromato-graphy The ESI-MS-determined molecular mass,of
28 114.6 ± 1.2 Da,was lower than that calculated from the sequence (28 246.41 Da),the difference (131.9 Da) accounting for the missing N-terminal Met in the His-tagged recombinant enzyme The specific activity of the native and His-tagged UMP kinase under optimal assay
protein,respectively,which correspond to molar activities
UMP kinase,which can be stored at room temperature for
increased the thermal stability of B subtilis UMP kinase,
nucleotide The protective effect of UTP against thermal denaturation is specific for this nucleotide,ATP and GTP being ineffective UTP was demonstrated also to increase the thermal stability of E coli UMP kinase [9,10]
The single Cys residue (Cys206) of UMP kinase from B subtilis is conserved in the enzyme from
S aureus, Pseudomonas aeruginosa, Bordetella pertussis, Mycobacterium tuberculosis, Neisseria meningitidis,
UMP kinase from E coli, Sal typhi, Yersinia pestis or Haemophilus influenzae This residue reacted with DTNB under native conditions The kinetics was fitted to a single-exponential equation except for an initial missing amplitude, which corresponds probably to the reaction with DTNB of the partially unfolded fraction of enzyme (Fig 3) The kobs
Fig 2 Modelled structure of UMP kinase from Bacillus subtilis One
monomer is shown in CPK representation Colours (from blue to
violet) indicate residue conservation (weakly to strongly) as computed
by CONSURF [35] and visualized using RASMOL (http://www.umass.edu/
microbio/rasmol/) The second monomer in the model is shown as
green ribbon ATP molecules are shown in yellow.
Trang 67· 10)3Æs)1(in its absence) GTP,ATP and Mg2+did not
significantly affect the reaction-rate constant of UMP
UTP,reverses its protective effect
The fluorescence spectrum of UMP kinase from B
348 nm,indicating that Trp58 is fully exposed to the
solvent UTP shifted the fluorescence maximum to 331 nm,
reversed this effect Binding of UTP to the enzyme exhibits a
slight cooperative effect (Hill number 1.5, Kd22 lM) GTP
binding,which decreased the fluorescence intensity at
Kinetic and nucleotide-binding properties
The UMP kinase activity with various nucleoside
unusually low specific activities for this class of enzymes
The maximal rate was found with ATP and dATP When
NTPs were used in the mixture,the highest specific activity
was obtained with ATP + GTP,indicating a requirement
for GTP for expression of full catalytic activity (Table 1)
When the ATP concentration was varied in the presence
concentrations of UMP was dependent on GTP Thus,in
increases in UMP In the presence of GTP,saturation was
mono-phosphate GTP showed the most important activating
effect,the half-maximum activation being reached at
Esterification of 3¢-OH in dGTP with the anthraniloyl group increased,by one order of magnitude,the affinity of the parent nucleotide for B subtilis UMP kinase ITP was
a rather weak activator,whereas GMP was ineffective (Fig 4A) These results are significantly different from those obtained with E coli UMP kinase,in which GMP,cGMP and even guanosine exerted activation UTP antagonized the activation by GTP (Fig 4B) In the absence of GTP,
UTP at which half-inhibition was observed Of the nucle-oside monophosphates tested,5F-UMP and 6-aza-UMP showed the most interesting properties Both analogues were actively phosphorylated by UMP kinase from B sub-tilisand E coli (Table 2),which is in line with the cytotoxic effect of the corresponding nucleosides on these organisms [42,43]
Kinetic and intrinsic fluorescence studies of UMP kinase from B subtilis suggested that the allosteric effectors,GTP and UTP,bind to identical or largely overlapping sites However,in the absence of binding studies,ambiguities,as regards the identity of the activating or inhibitory sites, still persist Therefore,we explored several analogues of UTP and GTP capable of binding to allosteric site(s) of UMP kinase and giving an appropriate fluorescence signal Ant-dGTP,which activated UMP kinase with a higher affinity than that of the parent nucleotide,exhibited strong fluorescent properties in aqueous solution upon excitation
at 330 nm with a maximum at 425 nm The addition of enzyme in excess over Ant-dGTP increased its fluorescence intensity by more than threefold UTP and GTP,and,to a lesser extent,the corresponding nucleoside diphosphates, displaced the fluorescent analogue from UMP kinase Under identical experimental conditions,ATP competed weakly with Ant-dGTP binding,whereas UMP and GMP were ineffective (Fig 4C) Ant-dUTP behaved similarly, although the enhancement in fluorescence intensity of the analogue,upon addition of the enzyme,was lower (by a factor of 1.8) Both GTP and UTP competed effectively with Ant-dUTP,whereas ATP was almost completely ineffective (data not shown)
Fig 3 Reaction of UMP kinase from Bacillus subtilis with
5,5¢-di-thiobis (2-nitrobenzoic acid) (DTNB) under native conditions Enzyme
(25 l M in terms of monomer),in 50 m M Tris/HCl (pH 8) containing
100 m M NaCl,was thermostated at 25 C and treated with DTNB
(0.2 m M final concentration) The absorbance (A) was then read at
412 nm for 20 min The molar ratio of thiol reacted was calculated
using the mass of His-tagged protein of 28.1 kDa,and e of
thio-nitrobenzoate anion of 13.6Æm M )1
Table 1 Reaction rate of UMP kinase from Bacillus subtilis with vari-ous nucleoside triphosphates (NTPs) The reaction medium (0.5 mL final volume) was the same as that described in the legend to Fig 4 The concentrations of NTPs and of UMP were constant (1 m M ) The reaction rate with ATP + GTP (14.2 lmolÆmin)1Æmg)1of protein) is considered to be 100%.
NTP Reaction rate (%)
Trang 7Bacterial UMP kinases are the most intriguing members of the NMP kinase family,for several reasons These enzymes
do not exhibit any sequence homology with other NMP kinases described so far,exist in oligomeric form (hexamers) and are subject to complex regulation by nucleotides In addition,the pyrH gene product from enteric bacteria directly participates in the pyrimidine-specific control of the
systematic analysis of the pyrH gene product in different bacteria with the aim of correlating differences in enzyme structure with substrate specificity,stability and the capa-bility of organisms to adapt to environmental conditions In this respect,UMP kinases from E coli (a Gram-negative bacterium) and B subtilis (a Gram-positive bacterium) have some characteristics in common,i.e high sequence identity, identical quaternary structure and similar catalytic proper-ties,such as activation by GTP and inhibition by UTP They also exhibit significant differences in stability and physicochemical properties that might be rationalized in structural terms [38]
According to the current modelling study,UMP kinase from B subtilis is made of one domain comprising a central,mostly parallel,b-sheet surrounded by a-helices The N-terminus (residues 5–142) of protein would also build up the dimer interface The position of ATP in the model was deduced from the position of the AMP-PNP (adenosine b,c-imido-5¢-triphosphate) and that of ADP in the crystal structures of NAGKec (PDB1gs5) and of CK-like CPSpf (PDB1e19),respectively It suggests that the C-terminus (residues 143–240) is probably the phos-phate donor-binding site The UMP- and the allosteric binding sites remain to be identified Our model suggested that Trp58 in the B subtilis UMP kinase is 10 A˚ from the active site and close to a solvent-exposed groove The latter is formed by the helix a2 (residues 55–67), the following loop (68–74) and a second loop (137–141) The equivalent region in the E coli UMP kinase contains several conserved residues whose substitutions (Arg62His, Asp77Asn) affect the kinase activity and especially its allosteric regulation [12] The fluorescence of Trp119 in the E coli UMP kinase,modelled in the same region,is also affected by allosteric effectors [9] The corresponding region (residues 100–150) of the related glutamate-5-kinase from E coli also contained an allosteric site according to two characterized mutations [45] The unique cysteine (Cys206) of B subtilis UMP kinase was assigned at the C-terminus of helix a7 facing the strand b8 The reactivity
Fig 4 Interaction of UMP kinase from Bacillus subtilis with various
nucleotides acting as allosteric effectors The reaction medium (0.5 mL
final volume) buffered with 50 m M Tris/HCl (pH 7.4) contained 50 m M
KCl,2 m M MgCl 2 , 1 m M phosphoenolpyruvate,0.2 m M NADH,
2 m M ATP,1 m M UMP and different concentrations of GTP,dGTP,
3¢-anthraniloyl-2¢-deoxyguanosine-5¢-triphosphate (Ant-dGTP),ITP
or GMP,as indicated,and 2 units of each of pyruvate kinase,NDP
kinase and lactate dehydrogenase The reaction was started with pure
UMP kinase (Top) Ant-dGTP (h); GTP (j); dGTP (m); ITP (s);
GMP (d) (Middle) The variable nucleotide was UTP,in the presence
of constant concentrations of GTP: 0.1 m M (j); 0.2 m M (m); 0.5 m M
(h) (Bottom) Binding of Ant-dGTP (2 l M ) to UMP kinase of
B subtilis (20 l M ) as determined at 420 nm (excitation k at 330 nm)
and displacement by UTP (d),GTP (j),ATP (h) and GMP (n).
Table 2 Comparative kinetic parameters of UMP kinase from Bacillus subtilis and Escherichia coli with UMP analogues The reaction medium is the same as that described in the legend to Fig 4 The concentrations of ATP (2 m M ) and GTP 0.5 (mM) were constant The concentration of UMP and of its analogues varied between 0.02 and 2 m M V m is expressed as lmolÆmin)1Æmg)1of protein 5F-UMP,5-fluoro-UMP.
NMP
V m K m (l M ) Relative V m /K m V m K m (l M ) Relative V m /K m
Trang 8to DTNB is affected specifically by UTP This effect
comes in line with the effect of UTP on the protein
stability,as the flexibility decrease would maintain the
buried Cys206 as almost completely inaccessible
Another striking characteristic of B subtilis UMP kinase
is its high requirement for GTP for full catalytic activity,
an effect antagonized by UTP This property is shared by
UMP kinase from other Gram-positive organisms such as
M Straut,unpublished data) At physiological pH,GTP
activates UMP kinase from E coli or H influenzae by only
three- to fourfold Binding studies with dGTP and
Ant-dUTP,and displacement by the natural effectors GTP and
UTP, suggest that the activator and inhibitor sites are
identical Although chemical modification experiments on
and UTP sites overlap [9],direct proof that the allosteric
effectors compete for the same site in bacterial UMP kinases
was still missing We propose,by analogy with aspartate
transcarbamylase,which binds at the same regulatory-site
CTP (inhibitor) and ATP (activator) [46],that UMP kinase
requires essentially the same residues for interacting with
both UTP and GTP The redistribution of hydrogen bond
network upon binding of closely related nucleotides has
been previously shown with other enzymatic systems [15] It
remains to be demonstrated that the opposite effects on
conformation and catalysis,accompanying the binding of
GTP and UTP,result primarily from interactions at the
level of the heterocycle
Whatever the mechanism of activation may be under
of GTP and UTP play a role in the metabolism of B subtilis
in vivo? From the total concentration of different NTPs and
Mg2+in B subtilis,[47,48],it is conceivable that GTP is the
major player besides the two substrates ATP and UMP,as
form of the nucleotide in the bacterial cell,is only a weak
from B subtilis in vivo,via the allosteric site This picture is
different for UMP kinase from Gram-negative organisms,
nanomolar range [9] (Gilles et al unpublished results)
A last point,worthy of mention,is the involvement of
UMP kinase in the metabolism of cytotoxic nucleobases or
nucleosides,which act as phosphorylated derivatives
inter-fering either with DNA or RNA synthesis or by inhibiting
key enzymes in the formation of nucleoside triphosphates
[42,43] In this respect, both 5F-uridine and 6-azauridine are
readily phosphorylated to 5F-UMP and 6-aza-UMP by
bacterial uridine kinase UMP kinase from B subtilis and
contributing,with the nonspecific NDP kinase,to the
formation of the highly toxic compounds F-UTP and
6-aza-UTP
Acknowledgements
We thank Jan Neuhard for many inspiring discussions,Nicolas
Glansdorff for the kind gift of E coli 14:40-42,Sylvie Pochet for
help in the synthesis of 5F-UMP and 6-Aza-UMP,Hiroshi
Sakamoto and Tudor Borza for cloning and complementation experiments,Jean-Claude Rousselle for mass spectrometry measure-ments,Dominique Douguet and Susan Michelson for constructive criticism,He´le`ne Munier-Lehmann for fruitful comments,and Re´gine Lambrecht for excellent secretarial help This work was supported by grants from the Centre National de la Recherche Scientifique (URA2185),Institut Pasteur (AC02) and AstraZeneca R
& D, Boston, Inc.
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