Eriksson, Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Box 575, Biomedical Center, S-751 23 Uppsala, Sweden Fax: +46 18550762 Tel: +46
Trang 1parvum UMP kinase – a potential antibacterial drug target Louise Egeblad-Welin1, Martin Welin2,*, Liya Wang1 and Staffan Eriksson1
1 Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Sweden
2 Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Sweden
Ureaplasma parvum belongs to the class Mollicutes,
which have the smallest genomes known in any
free-living organisms, and a very low G + C content [1] It
is a human pathogen that normally colonizes the
urogenital tract, where it is involved in a variety of
diseases such as urethritis and prostatitis During
pregnancy, it is an opportunistic pathogen and can
cause spontaneous abortions and premature birth The
bacteria can be transferred vertically from mother to
child during birth, and give rise to meningitis and
pneumoniae in newborns [2]
U parvum uridine monophosphate kinase (UpUMPK) (EC 2.7.4.22), coded by the PyrH gene, catalyses the reversible phosphorylation of uridine monophosphate (UMP) using a nucleoside triphosphate (NTP) as phosphate donor [3,4] It has been cloned and the recombinant enzyme characterized [4] UpUMPK has high sequence identity to other UMPKs from bacte-ria and archaea (Fig 1); the sequence identity to
UMP-Ks from Escherichia coli, Pyrococcus furiosus, Sulfolobus solfataricus, Haemophilus influenzae and Streptococcus pneumoniaeis 34, 26, 25, 34 and 38%, respectively The
Keywords
bacterial UMP kinase; Mollicutes;
mycoplasma; subunit interaction;
Ureaplasma parvum
Correspondence
S Eriksson, Department of Anatomy,
Physiology and Biochemistry, Swedish
University of Agricultural Sciences, Box 575,
Biomedical Center, S-751 23 Uppsala,
Sweden
Fax: +46 18550762
Tel: +46 184714187
Email: Staffan.Eriksson@afb.slu.se
*Present address
Structural Genomics Consortium, Karolinska
Institutet, Stockholm, Sweden
Database
The structure has been submitted to the
Protein Data Bank under the accession
number 2va1
(Received 29 June 2007, revised 19 October
2007, accepted 22 October 2007)
doi:10.1111/j.1742-4658.2007.06157.x
The crystal structure of uridine monophosphate kinase (UMP kinase, UMPK) from the opportunistic pathogen Ureaplasma parvum was deter-mined and showed similar three-dimensional fold as other bacterial and archaeal UMPKs that all belong to the amino acid kinase family Recom-binant UpUMPK exhibited Michaelis–Menten kinetics with UMP, with Km and Vmax values of 214 ± 4 lm and 262 ± 24 lmolÆmin)1Æmg)1, respec-tively, but with ATP as variable substrate the kinetic analysis showed posi-tive cooperativity, with an n value of 1.5 ± 0.1 The end-product UTP was
a competitive inhibitor against UMP and a noncompetitive inhibitor towards ATP Unlike UMPKs from other bacteria, which are activated by GTP, GTP had no detectable effect on UpUMPK activity An attempt to create a GTP-activated enzyme was made using site-directed mutagenesis The mutant enzyme F133N (F133 corresponds to the residue in Escherichia coli that is involved in GTP activation), with F133A as a control, were expressed, purified and characterized Both enzymes exhibited negative coo-perativity with UMP, and GTP had no effect on enzyme activity, demon-strating that F133 is involved in subunit interactions but apparently not in GTP activation The physiological role of UpUMPK in bacterial nucleic acid synthesis and its potential as target for development of antimicrobial agents are discussed
Abbreviations
NDPK, nucleoside diphosphate kinase; UMPK, uridine monophosphate kinase.
Trang 2primary sequence and crystal structures of E coli
UMPK and the archaea P furiosus and S solfataricus
UMPKs showed that these enzymes belong to the amino
acid kinase family [5–8] In eukaryotic cells, the
corre-sponding enzyme is CMP-UMPK (EC 2.7.4.14), which
is a member of the nucleoside monophosphate kinase
family [9]
Studies with Mycoplasma genitalium using
transpo-son mutagenesis showed that UMPK is essential for
the survival of the organism [10,11] The UMPKs
(PyrH genes) of E coli, H influenzae and St
pneumo-niae have also been shown to be essential [12–14]
Therefore, UMPK is a potential drug target for the
development of antimicrobial agents, and it is of great
importance to study the structure and function of these enzymes
E coli, Bacillus subtilis and St pneumoniae UMPKs are hexamers and are activated by GTP, inhibited by UTP, and show Michaelis–Menten kinetics with UMP Furthermore, St pneumoniae UMPK displays positive cooperativity with ATP [5,14–16], as was also recently shown with the enzymes from other Gram-positive bacteria, e.g B subtilis and Staphylococcus aureus [17] In E coli, UMPK residues Arg62, Asp77, Thr138 and Asn140 have been suggested to be involved in the interaction with GTP, as mutation of these residues abolished the GTP activation [6,18] However, S solfataricus UMPK is not activated by
Bacteria specific loop
Archaea specific loop
Fig 1 Sequence alignment of UMPKs Accession numbers: H influenzae, P43890; E coli, P0A7E9; N meningitidis, P65931; St pneumo-niae, Q97R83; Streptococcus pyogenes, P65938; B subtilis, O31749; U parvum, Q9PPX6; S solfataricus, Q97ZE2; P furiosus, Q8U122 Secondary elements for U parvum UMPK are listed above the alignments Completely conserved residues are colored in red and similar res-idues in yellow.
Trang 3GTP, which was previously suggested to be specific
to archaeal UMPKs [8]
In this study, recombinant UMPK from U parvum
was enzymatically characterized, particular with regard
to the substrates UMP and ATP, the inhibitor UTP
and the potential activator GTP The crystal structure
was determined by X-ray crystallography in complex
with a phosphate ion A cross-talk region between two
subunits of UpUMPK was identified, which
corre-sponded to the region in E coli UMPK that contains
the key residues Thr138 and Asn140 that are involved
in GTP activation Residue Phe133 of UpUMPK
(cor-responding to Asn140 in E coli UMPK, Fig 1) was
mutated to either Asn or Ala, and the resulting mutant
enzymes were characterized
Results
Overall structure
The structure of the UpUMPK was determined by
X-ray crystallography to a resolution of 2.5 A˚ with a
final R value of 23.3% and Rfree of 28.5% (Table 1)
The enzyme is a hexamer composed of three dimers
that are related by threefold symmetry (Fig 2A) The
monomer subunit consists of an a⁄ b-fold with a
nine-stranded twisted b-sheet surrounded by eight a-helices
and one 310 helix (Fig 2B) The monomers are simi-lar, and when each subunit (B, C, D, E and F) is superpositioned on subunit A, the rmsd varies between 0.169 and 0.419 A˚ A flexible loop between b5 and b6 (amino acids 166–175) can only be traced in the elec-tron density for the E subunit, with B factors of approximately 50 A˚2 In UMPK from P furiosus, this loop is responsible for binding the adenine base of an ATP analogue (Protein Data Bank accession number
Table 1 Data collection and refinement statistics Values in
paren-theses refer to the data in the highest-resolution shell ESRF,
European synchrotron radiation facility.
b, 96.6
c, 96.3
b, 105.8
rmsds
α2
A
B
α3
α1
α8 α6
α7 α5
α4 β4
β2 β1
β6
β9 β8 β7
β5
η1 β3
Fig 2 (A) UpUMPK as a hexamer; a phosphate ion is seen in every monomer The dimeric couples A + B, C + D and E + F are colored green, pink and blue, respectively (B) The monomer of UpUMPK
in complex with a phosphate ion The flexible loop that was only observed in one subunit is colored in orange.
Trang 42BRI), and, as no ATP⁄ ATP analogue is bound to the
enzyme, the loop is not held in a tight position The
forces that hold the hexamer together are (a)
hydro-phobic interactions between the dimeric couples
(A + B, C + D and E + F), (b) a few hydrogen
bonds, and a hydrophobic interaction between A + C,
B + E and D + F, and (c) electrostatic forces in the
central channel of the hexamer between B, C and F,
and A, D and E The hydrophobic interactions
between A and B (Fig 3A,B) are formed between the
antiparallel a3-helices from each subunit, primarily by
Leu, Met and Ile One hydrogen bond could be
identi-fied at each end of the interacting a-helices between
Asn86 and the carbonyl carbon of Leu62 Between A
and C, the a7 from each subunit is connected via
two hydrogen bonds between Thr197 and Glu204
(Fig 3A,C), and a hydrophobic interaction between
Thr131 and Phe133 (Fig 3D) The central channel of
the hexamer is made up of two layers of electrostatic
forces on top of each other, with one layer rotated by
60 The amino acids found in the electrostatic hole in
each layer are Lys102 and Asp104 Lys102 is held in
position by Asp104, and, in the interaction between A,
D and E, a water molecule is hydrogen-bonded to the
three lysines (Fig 3E) There is no water molecule
fix-ing the three lysines from the B, C and F subunits,
and there is no direct interaction between subunits A
and F, B and D, or C and E
A phosphate ion was found in the donor site of all
subunits (Fig 2B), although the protein was
crystal-lized in the presence of 5 mm GTP The B factors for
the phosphate ions in the subunits varied from 46–
55 A˚2 Structural alignments with UMPK from E coli
(Protein Data Bank accession numbers 2BND and
2BNF) indicated that the phosphate ion in UpUMPK
was bound in the position corresponding to the
b-phosphate of either UDP or UTP Combinations of
co-crystallization and soaking were performed in
order to bind either substrate or inhibitor However,
in all collected data sets (UpUMPK co-crystallized
with UMP, and UTP or with no added ligand), a
phosphate ion was detected in the active site This
indicates that phosphate ions were bound to the
enzyme during expression and purification, since no
phosphate buffers were used during the preparation
procedures
As no structural data for UpUMPK with UMP
bound were obtained, the binding of UMP to the
active site was modeled by structural alignment with
UMPK from E coli with UMP bound [6], which gave
an rmsd of 1.29 A˚ for 212 Ca-atoms The binding of
UMP in E coli creates a tightly closed conformation
with a2 (Fig 4A) In UpUMPK, this a-helix has a
more open conformation in the absence of UMP (Fig 4A) The amino acid residues responsible for binding of UMP are relatively conserved (Fig 4B); the only difference is a Phe133 found in the position corre-sponding to Asn140 in E coli The probable binding motif for the uracil base is through hydrogen bonds from N3 to the backbone O of Phe133, and from O4
to the backbone N and the side chain of Thr131, with the ribose moiety anchored by two hydrogen bonds, one from 2¢-OH to the side chain of Asp70, and one from the backbone N of Gly63, and the phosphate group forming hydrogen bonds from O1 to Arg55, from O2 to the backbone N and the side chain of Thr138, and from O3 to the backbone N of Gly50 A P-loop (GXXXXGKS⁄ T) that is usually found in nucleotide binding enzymes is not present in UpUMPK [19] UpUMPK contains instead a glycine-rich motif within amino acids 44–54 that is responsible for bind-ing of the phosphate ion These amino acids are rela-tively conserved among the UMPKs, with an amino acid sequence motif as follows: V⁄ IXV ⁄ IXGGGXXXR (Fig 1)
Functional characterization The substrate specificity of purified recombinant UpUMPK was explored using a coupled spectrophoto-metric assay [20], and several ribonucleoside mono-phoshates and deoxyribonucleoside monophosphates were tested as phosphate acceptors with ATP as phos-phate donor The only effective acceptor was found to
be UMP, and the pH optimum of the reaction was 6.8 It was also observed that a stoichiometry between
Mg2+ and ATP of 2 : 1 gave 1.6-fold higher catalytic rates compared to a 1 : 1 stoichiometry (data not shown) Therefore, the Mg2+:ATP ratio was kept at
2 : 1 in all further experiments, in analogy with other UMPK studies [14,17]
Two-substrate kinetics with UMP and ATP Initial two-substrate kinetic assays were performed with varying UMP concentrations (100–2000 lm) and various fixed ATP concentrations (100, 200, 500 and
1000 lm), and the dependency of the velocity on sub-strate concentration was hyperbolic for UMP as the varied substrate (Fig 5A) We then calculated what should be the true Km and Vmax values for UMP, giving 214 ± 4 lm and 262 ± 24 lmolÆmin)1Æmg)1, respectively
However, with ATP as the variable substrate, the kinetic curves showed a detectable deviation from Michaelis–Menten kinetics, especially at low substrate
Trang 5concentrations (Fig 5B) The best fit was therefore to
the Hill equation, giving an n value of 1.54 ± 0.10,
demonstrating positive cooperativity with ATP With
1 mm UMP, the K0.5,app(ATP) was 316 ± 54 lm and
the Vmax,app(ATP) was 172 ± 23 lmolÆmin)1Æmg)1,
which is similar to the values calculated in the initial
kinetic analysis
UTP as end-product inhibitor The nature of UTP inhibition was investigated in assays with fixed ATP (1 mm) and variable UMP con-centrations (50–1000 lm) Double-reciprocal plots at various UTP concentrations demonstrated that UTP was a competitive inhibitor towards UMP, with a Ki
C
B A
A
B
E204
E204
T197
T197
A
C
F133
T131 A
C
D
E
A
K102 D104
K102
D104
D104 K102
C
E
D
Fig 3 (A) Interactions between subunits A, B and C (B) Hydrophobic interaction between the a4 helices from A and B (C) Interaction between a7 helices from A and C Hydrogen bonds form between Thr197 and Glu204 (D) Hydrophobic interaction between A and C, formed
by Thr131 and Phe133 (also referred to as the cross-talk region) (E) Electrostatic interactions in the central channel of the enzyme between subunits A, D and E Asp104 holds Lys102 in position in each subunit A water molecule is fixed by the three lysines from each subunit.
Trang 6value of 0.7 mm (Fig 6A) When ATP was the
vari-able (50–1000 lm) substrate at a fixed UMP
concentra-tion (1 mm), the inhibiconcentra-tion by UTP affected primarily
the Vmax values, while the K0.5⁄ Km values at various
UTP concentrations were in the same range Thus,
UTP inhibition was noncompetitive towards ATP,
with a Ki value of 1.2 mm (Fig 6B) When this data
set was fitted to the Hill equation, the n values were
1.4, 0.98 and 1.0 for UTP at 0, 0.5 and 1.0 mm,
respec-tively, which indicates that the positive cooperativity
behavior with ATP is altered by the presence of UTP
Determination of enzyme-bound orthophosphate
and inhibition of enzyme activity by
orthophosphate
In the UpUMPK structure, a phosphate ion was found
in the active site, and this raised a question concerning
the actual orthophosphate content in the enzyme used
in the functional studies Therefore, the phosphate
content in the enzyme preparation was determined using a colorimetric method A concentrated enzyme solution was precipitated with 5% perchloric acid at low temperature to release bound phosphate ions The concentration of free phosphate in the supernatant was 3.52 lm, and the total concentration of enzyme was
A
B
F133
N140
D70
G56
R55 G50
T138
T131
T138
T145
R62 G57
G63 D77
Fig 4 (A) Superposition of UpUMPK (green) on top of E coli
UMPK (blue) at the active site (B) Binding of UMP to E coli UMPK
and the location of amino acid residues in UpUMPK based on the
superposition.
A
B
C
Fig 5 (A) Activity versus [UMP] at various concentrations of ATP (lM) (B) Activity versus [ATP] at various concentrations of UMP (lM) (C) Lineweaver–Burk plot of 1 ⁄ v against 1 ⁄ [UMP] at various concentrations of ATP (lM).
Trang 791.12 lm, giving a molar ratio of UpUMPK⁄
phos-phate of 25⁄ 1
The effect of orthophosphate on enzyme activity
was examined It was shown that phosphate inhibited
UpUMPK activity with an IC50 value of 1 mm
(Fig 7)
Functional consequences of F133N and F133A
mutations
GTP is an activator for all bacterial UMPKs studied
to date [5,14–17], and it was therefore tested with
UpUMPK In an assay with 1 mm UMP and ATP, the
addition of 0.5 or 1 mm GTP resulted in no detectable
change in UpUMPK activity
In order to find an explanation for the lack of GTP
activation, the UpUMPK structure was compared to
that of E coli UMPK In E coli UMPK, residue
Asn140 forms a hydrogen bond to Thr138 of a
neigh-boring subunit (Fig 8) The backbone of Asn140 and
the side chain of Thr138 also form hydrogen bonds to the uracil base Mutations of either Thr138 or Asn140
to Ala abolished GTP activation, indicating that these residues are involved in GTP activation of the E coli UMPK [6] In UpUMPK, a region between subunits A and C had a Phe133 in the position corresponding to Asn140 in the cross-talk region of E coli UMPK Phe133 is not able to form hydrogen bonds due to its hydrophobic interactions with Thr131 (Fig 3D)
To mimic E coli UMPK, Phe133 of UpUMPK was mutated to Asn or Ala The mutant enzymes, F133N and F133A, were expressed, purified and characterized With 1 mm UMP and ATP as substrates, the activities
of F133N and F133A were only 50 and 20% of that
of the wild-type enzyme Similar to the situation with wild-type UpUMPK, addition of 0.5 and 1 mm GTP resulted in no detectable activation of F133N or F133A mutant enzymes
A
B
Fig 6 (A) UTP acts as a competitive inhibitor towards UMP.
Double-reciprocal plot of 1 ⁄ v versus 1 ⁄ [UMP] at 0, 0.1 and 0.5 mM
UTP (B) UTP acts as a noncompetitive inhibitor towards ATP.
Activity versus [UMP] at 0, 0.5 and 1 mM UTP.
0 50 100 150 200
[Pi] µ M
Fig 7 Inhibition of UpUMPK activity by Pi [UMP] and [ATP] ¼ 1 mM.
C N140
T138
N140
T138
Fig 8 Cross-talk region of E coli UMPK between subunits A and
C, with UMP bound to the active site Amino acid residues T138 and N140 are found in the cross-talk region (Protein Data Bank accession number 2BNE) [6].
Trang 8With variable UMP concentration and fixed ATP
concentration, both the F133N and F133A mutants
dis-played negative cooperativity, and the Hill coefficients
were 0.65 ± 0.05 and 0.85 ± 0.05, respectively (Fig 9)
At 1 mm ATP, the K0.5,app(UMP) and Vmax,app values
for F133N were 1100 ± 150 lm and 107 ± 15 lmolÆ
min)1Æmg)1, respectively For F133A, the K0.5,app(UMP)
and Vmax,app values were 896 ± 212 lm and 63 ±
2 lmolÆmin)1Æmg)1, respectively The K0.5,app (UMP)
values for the mutant enzymes were four- to fivefold
higher than that of the wild-type enzyme The Vmax,app
values were also affected; they were twofold lower in
case of F133N, and approximately fourfold lower for
the F133A mutant
Discussion
In this study, we have investigated UpUMPK and
shown that the structure resembles UMPKs from
bac-teria and archaea belonging to the amino acid kinase
family A phosphate ion was bound to all subunits in
the enzyme However, the molar content of
orthophos-phate in the soluble UMPK was only 4% The
concen-tration of phosphate giving 50% inhibition of enzyme
activity (IC50 value) was 1 mm, indicating that the
phosphate did not have a very high affinity for the
enzyme The discrepancy between the structural and
functional results is not easily explained and may be
methodological At present, we cannot distinguish the
possibilities that the enzyme contains tightly bound
phosphate ions that cannot be released by acid
precipi-tation, or alternatively that only the phosphate-binding
fraction of the enzyme can form crystals
The Km value for UpUMPK with UMP is high
(214 ± 4 lm) compared to the Km values for other
UMPKs, e.g S solfataricus, 14 lm; E coli, 43 lm (at
pH 7.4); B subtilis, 30 lm; St pneumoniae, 100 lm [8,14–16] A possible reason for the high Kmvalue for UpUMPK with UMP could be the presence of a phos-phate ion in the active site However, as discussed above, the kinetic results most likely reflect the proper-ties of the native fully active UpUMPK enzyme Positive cooperativity with ATP was observed when the assays were performed with ATP as the variable substrate (n value of 1.5) However, in the inhibition experiment with UTP, the n values were close to 1.0, indicating that the presence of UTP abolished the positive cooperativity observed with ATP alone At present, there is no clearcut explanation for this obser-vation Nevertheless, the cooperative behavior of UpUMPK with varied ATP concentrations is less pro-nounced than that reported with other Gram-positive bacterial UMPKs (n values of 1.9–2.5 with 1 mm UMP) [14,17]
The fact that UTP is a competitive inhibitor for UMP
is in agreement with the structural data from E coli UMPK, where it has been shown that UTP binds to the base moiety in the active site [6] For S solfataricus, the same pattern was observed with UTP and UMP, but in that case UTP is a competitive inhibitor towards ATP [8] The Ki values for UTP versus UMP (0.7 mm) and ATP (1.2 mm) are high, and may indicate that UTP is
an inefficient inhibitor in vivo
UMPKs from E coli, Salmonella typhimurium, H in-fluenzae, Neisseria meningitidis, B subtilis, St pneumo-niae, Staphylococcus aureus and Enterococcus faecalis were all activated by GTP by a factor of 2.5–18.5 [17] The UMPK from the archaea S solfataricus was the first UMPK for which lack of activation by GTP was shown [8], and in this study we have shown that UMPK from U parvum also lacks activation by GTP
The mutational study of residue F133 was per-formed to clarify whether this residue is involved in GTP activation F133 was mutated to Asn in an attempt to create a GTP-activated enzyme, and F133A was prepared and tested as a control Neither of the UpUMPK mutants F133N and F133A were activated
by GTP Thus, this residue alone is not responsible for GTP activation However, an interesting feature was observed UpUMPK F133N exhibited negative cooper-ativity with UMP as a substrate (n value of 0.65), and the same was true of UpUMPK F133A to a lesser extent (n value of 0.85) This is the first time that nega-tive cooperativity has been described with a bacterial UMPK The observed negative cooperativity may be explained by alteration of the geometry of the active site in the neighboring subunit when UMP binds to the enzyme, i.e mutation of residue F133, which is
F133A F133N
60
50
40
30
20
10
0
[UMP]/µ M
800 1000 1200
Fig 9 Substrate saturation curves of UpUMPK mutant enzymes:
F133N and F133A with UMP as variable substrate.
Trang 9located in the interface of two subunits, may have
affected the mode of subunit interaction
Jensen et al (2007) have compared the sequence and
structure of S solfataricus UMPK to those of the
known bacterial UMPKs A loop between a6 and a7
was only present in archaea, and was referred to as the
archaea-specific loop (Fig 1) Another difference that
was detected was in the loop between a3 and a4,
which was absent in archaea, and is therefore referred
to as the bacteria-specific loop (Fig 1) Jensen et al
(2007) suggested that either the amino acid residues
found in the bacteria-specific loop or the lack of a
known nucleotide binding motif GXXGXG [21] in the
N-terminus was responsible for the lack of GTP
acti-vation [8] A comparison of UMPKs from U parvum,
E coli and S solfataricus, chosen to represent
myco-plasma, bacteria and archaea, showed that they all
shared the same fold As UpUMPK has essentially the
same fold as E coli UMPK, it is unlikely that residues
in the bacteria-specific loop are involved in the
activa-tion However, the N-terminal GXXGXG motif is not
found in UpUMPK, suggesting that this motif may be
involved in GTP activation
UMPK is involved in both de novo and salvage
syn-thesis of DNA and RNA precursors The results
pre-sented here suggest that UpUMPK is an enzyme that is
mainly regulated by the UMP and orthophosphate
lev-els, and is not very sensitive to feedback inhibition by
the end-product UTP Furthermore, there is no
evi-dence for allosteric activation by GTP, although the
overall structure is highly similar to the other bacterial
UMPKs The relative simplicity of the apparent
regula-tion of this structurally complex enzyme may be due to
its lifestyle, as it grows in the urinary tract where the
salvage of uridine and uracil may serve as a rich source
for UMP biosynthesis One of the goals of this
investi-gation was to evaluate whether UpUMPK is a
promis-ing new target for development of antibacterial agents
Bacterial UMPKs have no sequence or structural
homology to the human enzyme CMP–UMPK, which
makes them potential targets for drug development,
but, in the case of UpUMPK, a search for
non-nucleo-side⁄ nucleotide inhibitors may be more successful
Experimental procedures
Site-directed mutagenesis
The expression plasmid pET-14b-UpUMPK has been
described previously by Wang [4] The mutants
UpUMPK-F133N and UpUMPK-F133A were constructed by
site-directed mutagenesis using the plasmid pET-14b containing
cDNA for UMPK The F133N mutation was created using
the following primers: F133N-fw (5¢-GATTTTTGTGGCT GGAACAGGAAACCCATATTTTACAACTGATTCG) and F133N-rv (5¢-CGAATCAGTTGTAAAATATGGGT TTCCTGTTCCAGCCACAAAAAT), with the altered nucleotides shown in bold and underlined The F133A mutation was created using the following primers:
F133A-fw (5¢-GTGGCTGGAACAGGAGCGCCATATTTTACA ACTGATTCG) and F133A-rv (5¢-CGAATCAGTTGTAA AATATGGCGCTCCTGTTCCAGCCAC) The mutations were verified by DNA sequencing using the BigDye termi-nator cycle sequencing kit and the ABI PRISM 310 genetic analyzer (PE Applied Biosystems, Foster City, CA, USA)
Expression and purification of recombinant enzymes
Both wild-type UpUMPK and the mutant enzymes were expressed in E coli BL21 (DE3) in Luria–Bertani (LB) med-ium The enzymes were overexpressed by induction with iso-propyl-b-d-thiogalactoside (0.16 mm) overnight at 37C, and bacteria were harvested by centrifugation at 4600· g for 15 min at 4C The pellet was resuspended in buffer A, containing 50 mm Tris⁄ HCl (pH 7.5), 0.2 m KCl, 5 mm MgCl2and 0.2 mm phenylmethylsulfonyl fluoride The cells were then disrupted by sonication for 5 min with 5 s pulses, and thereafter centrifuged for 30 min at 4600· g at 4 C Purification was carried out at 4C The supernatant was applied to a metal affinity column (TALON resin, BD Bio-sciences Clontech, Palo Alto, CA, USA) using the gravity flow procedure The column was washed first with buffer B, containing 20 mm Tris⁄ HCl (pH 7.5) and 0.2 m KCl, and then with buffer B and 20 mm imidazole The protein was eluted with buffer B and 250 mm imidazole The purity of the protein was analyzed by SDS⁄ PAGE [22], and the pro-tein concentration was determined according to the method described by Bradford [23], with BSA as the standard pro-tein For wild-type UpUMPK, approximately 110 mg pure protein was obtained from a 1 L culture
Crystallization
The UMPK contained an N-terminal His-tag with the sequence MGSSHHHHHHSSGLVPRGSHM Crystals were grown by vapor diffusion, under conditions of 0.2 m ammonium fluoride and 20% (w⁄ v) poly(ethylene glycol) 3350 at 15C The enzyme concentration was 1.8 mgÆmL)1, and 5 mm GTP was added to the protein The protein and crystallization solution were mixed equally (2 lL of each) in a hanging drop
Data collection
The crystals were flash-frozen in liquid nitrogen, using mother solution with the addition of 15% (v⁄ v) poly(ethylene
Trang 10glycol) 400 as cryoprotectant, and data were collected at
ID14-eh4 at the European synchotron radiation facility
(ESRF), Grenoble, France The data were indexed, scaled
and merged using mosflm [24] and scala [25], and the
crys-tals were found to belong to the space group P21with a
sol-vent content of 48% The content of the asymmetric unit was
six monomers
Structure determination and refinement
The structure was solved by molecular replacement using
molrep[26], with the monomer of UMPK from H
influen-zae (Protein Data Bank accession number 2A1F) as the
search model Simulated annealing was performed in cns
[27], and further refinement was performed in refmac5
[28], during which noncrystallographic symmetry (NCS)
restraints were applied to residues 5–160 and 193–230 with
tight main-chain and medium side-chain restraints Model
building was carried out in o [29] and coot [30]
In chain A, residues 1 and 169–176 are missing; in
chain B, residues 167–172 are missing; in chain C, residues
1, 2 and 169–174are missing; in chain D, residues 168–174
are missing; in chain F, residues 1 and 168–175 are missing
All amino acid residues were present in the E chain, and
part of the His-tag was observed in the B chain A few
amino acid residues are found in disallowed regions in the
Ramachandran plot; these are A4, A165, A167, B3, B4 and
F165, all of which are found at either the beginning or the
end of a chain The structure has been deposited to Protein
Data Bank under the accession number 2va1 All figures
were created using pymol [31], and sequence alignments
were created using clustalw [32] and espript [33]
Determination of enzyme-bound orthophosphate
The presence of orthophosphate in UpUMPK was determined
by a colorimetric method [34] Briefly, the buffer used for
UpUMPK preparation was exchanged with water using
a PD-10 column (GE Healthcare, Uppsala, Sweden), and
then the protein concentration was determined To 2 mL
UpUMPK solution, perchloric acid was added to a final
con-centration of 5%, and the mixture was incubated on ice for
10 min The mixture was then centrifuged at 16 000· g for
15 min at 4C to remove precipitated protein The
superna-tant was neutralized with KOH, and incubated on ice for
15 min After centrifugation at 16 000 g for 20 min at 4C,
the supernatant was used in the colorimetric assay as described
previously [34] The concentration of orthophosphate was
3.52 lm, and the UpUMPK concentration was 91.12 lm
Enzyme assays
The UMPK activity was determined using a coupled
spec-trophotometric assay [20] with a Cary 3 spectrophotometer
(Varian Techtron, Mulgrave, Australia) at 37C The reaction medium (final volume 1 mL) contained 50 mm Tris⁄ HCl pH 6.8, 5 mm dithiothreitol, 0.5 mg mL)1 BSA,
1 mm phosphoenolpyruvate, 0.3 mm NADH and 4 lmolÆ min)1Æmg)1ÆmL)1of pyruvate kinase and lactate dehydroge-nase Nucleoside diphosphate kinase (NDPK) was not added, as this did not lead to a significant change in the rates determined, as observed by Fassy et al [14], and avoids the complication of potential UTP formation The coupling enzymes (pyruvate kinase and lactate dehydroge-nase) were tested with ADP and UDP, and ADP showed a rate that was > 20 times that of UDP
In order to determine the true Kmfor UMP and ATP, a two-substrate assay was performed at four concentrations
of UMP and ATP (100, 200, 500 and 1000 lm) In the GTP-activation experiments, the concentrations of UMP and ATP were kept at 1 mm In all experiments, MgCl2
concentration was kept in a stoichiometry of 2 : 1 towards NTP The enzyme concentration was 0.5 lg per assay for the wild-type and UpUMPK-F133N, and 1 lg per assay for UpUMPK-F133A The decrease in [NADH] was moni-tored at 340 nm
Analysis of kinetic data
Kinetic data were evaluated by nonlinear regression analysis using either the Michaelis–Menten equation v¼
VmaxÆ[S]⁄ (Km+ [S]), or the Hill equation v¼ VmaxÆ[S]n⁄ (Kn
0:5 + [S]n), where Km is the Michaelis constant, K0.5 is the value of the substrate concentration [S] where v¼ 0.5 Vmax, and n is the Hill coefficient If n¼ 1, there is no cooperativity, if n < 1 there is negative cooperativity, and
if n > 1 there is positive cooperativity One unit corre-sponds to 1 lmol min)1
The inhibition studies were analyzed using equations for competitive and noncompetitive inhibitors For competitive inhibition, the equation is v¼ VmaxÆ[S]⁄ (Km(1 + [I]⁄
Ki) + [S]), and for noncompetitive inhibition the equation
is v¼ VmaxÆ[S]⁄ (Km+ [S])(1 + [I]⁄ Ki) Kifor UTP towards ATP was determined using the secondary plot of slope versus [UTP]
Acknowledgements The authors wish to thank Andrea Hinas and Fredrik So¨derbom (Department of Molecular Biology, Swedish University of Agricultural Science) for help with muta-genesis, Hans Eklund (Department of Molecular Biol-ogy, Swedish University of Agricultural Science) for help with the structure determination, and Mark Harris (Department of Molecular Biology, Uppsala Univer-sity) for proof reading This work was supported by grants from the Swedish Research Council and the