(Received for publication, May 5, 1997, and in revised form, June 30, 1997)
From the Department of Biochemistry, Brandeis
University, Waltham, Massachusetts 02254 and ¶ Laboratory of
Microbial Structure and Function, Rocky Mountain Laboratories, NIAID,
National Institutes of Health, Hamilton, Montana 59840
Inosine 5-monophosphate dehydrogenase (IMPDH) is
the rate-limiting enzyme in de novo guanine nucleotide
biosynthesis. IMPDH converts IMP to xanthosine 5
-monophosphate with
concomitant conversion of NAD+ to NADH. All IMPDHs
characterized to date contain a 130-residue "subdomain" that
extends from an N-terminal loop of the
/
barrel domain. The role
of this subdomain is unknown. An IMPDH homolog has been cloned from
Borrelia burgdorferi, the causative agent of Lyme disease
(Margolis, N., Hogan, D., Tilly, K., and Rosa, P. A. (1994)
J. Bacteriol. 176, 6427-6432). This homolog has replaced the subdomain with a 50-residue segment of unrelated sequence. We have
expressed and characterized the B. burgdorferi IMPDH
homolog. This protein has IMPDH activity, which unequivocally
demonstrates that the subdomain is not required for catalytic activity.
The monovalent cation and dinucleotide binding sites of B. burgdorferi IMPDH are significantly different from those of human
IMPDH. Therefore, these sites are targets for the design of specific
inhibitors for B. burgdorferi IMPDH. Such inhibitors might
be new treatments for Lyme disease.
Inosine 5-monophosphate dehydrogenase catalyzes the conversion of
IMP to XMP1 with the
concomitant reduction of NAD to NADH. This reaction is the
rate-limiting step in guanine nucleotide biosynthesis, and is therefore
a target for numerous chemotherapeutic agents (1). IMPDH inhibitors are
used clinically in antiviral (ribavirin) and immunosuppressive
therapies (mycophenolate mofetil and mizoribine) (2-4). In addition,
IMPDH inhibitors have anti-tumor and antibiotic activity (5, 6).
Mammalian and bacterial IMPDHs have significantly different kinetic
properties and inhibitor sensitivities, which suggests that
species-specific IMPDH inhibitors can be developed that will be useful
in treating bacterial and parasitic infections (7-9). Indeed, many
studies have shown that purine metabolism plays an important role in
bacterial virulence (10-13).
The spirochete Borrelia burgdorferi is the causative agent of Lyme disease (14). This disease is transmitted by ticks of the Ixodes ricinus complex and is found worldwide. The genes encoding GMP synthase (guaA) and IMPDH (guaB) are located on a 26-kb circular plasmid (cp26) in B. burgdorferi (15). Genes carried by plasmids generally confer selective advantage in a particular environmental niche. The unique plasmid location of these housekeeping genes in B. burgdorferi may be related to their role in the transmission cycle between ticks and mammals. In ticks, guanine is the major nitrogenous waste product and therefore accumulates to high levels. However, in mammals, purine levels are low and limiting for bacterial growth. Therefore expression of the gua genes would be unnecessary for the survival of B. burgdorferi in ticks, but critical for survival in a mammalian host. Consistent with an adaptive role of cp26 in the mammalian environment, the gua genes are linked with ospC, which is induced during tick feeding. ospC encodes a protein that appears on the outer surface of the spirochete immediately preceding transmission to the mammal (16).
The identity of B. burgdorferi guaA was confirmed by complementation of GMP synthase-deficient Escherichia coli (15); however, the guaB homolog did not complement IMPDH-deficient E. coli. The failure to observe complementation by B. burgdorferi guaB most likely results from incompatible promoters or unstable protein. Alternatively, the guaB homolog may not encode an active IMPDH. Indeed, the guaB homolog, with a predicted molecular mass of 44 kDa, is 10 kDa smaller than typical IMPDHs. This difference in size results from the loss of 130 residues (residues 110-244, Chinese hamster IMPDH numbering) in the middle of IMPDH. These residues have been replaced with 50 residues of unrelated sequence (15).
The crystal structure of IMPDH provides little information about the
role of this missing region (17). IMPDH is an /
barrel protein,
with the active site located in the loops on the C-terminal ends of the
sheets. Residues 100-244 form an unusual insertion in a loop on
the N-terminal end of the
/
barrel. Therefore, this
"subdomain" is on the opposite end of the
/
barrel from the
active site. The subdomain is present in all of the IMPDHs characterized to date, although the subdomain of the guaB1
homolog from Mycobacterium leprae is missing 90 residues
(GenBankTM accession no. U00015). This region is also
absent in the homologous enzyme GMP reductase (18). A recent report
claims that activity is retained when the subdomain is deleted from
Chinese hamster IMPDH, although the experimental details were not
presented (17). The subdomain has no sequence or structural
similarities to proteins other than IMPDH. Thus the role of the
subdomain in IMPDH is unknown. Intriguingly, the subdomain is also
found in the guaB homolog from the relapsing fever
spirochete Borrelia
hermsii,2 which suggests
a species-specific modification of IMPDH in Lyme disease
spirochetes.
We have expressed the B. burgdorferi guaB homolog in E. coli. This protein has IMPDH activity, which demonstrates that the subdomain is not required for IMPDH activity. B. burgdorferi IMPDH has significantly different kinetic properties from human IMPDH. Therefore, B. burgdorferi IMPDH may be a target for the development of new treatments for Lyme disease.
IMP, NAD+, Trizma base, and
dithiothreitol were purchased from Sigma.
5-Ethynyl-1--D-ribofuranosylimidazole-4-carboxamide 5
-monophosphate was the generous gift of Dr. Akira Matsuda (Hokkaido University). Oligonucleotides were obtained from the Brandeis Oligonucleotide Facility.
Plasmid p68, containing the guaB gene of
B. burgdorferi B31, was previously isolated from a genomic
library constructed in the vector ZapII (Stratagene, La Jolla, CA)
(15). PCR was used to insert convenient restriction sites at the
beginning and end of the guaB gene. The following
oligonucleotides were used (mutations are underlined, restriction sites
are in bold lettering):
TGA-CTC-ATA(1)-TGC-CAA-ATA-AGA-TAA-CAA-AAG-AAG-CTT-TAA-C, inserts an NdeI site at the 5
end;
TGT-TCT-GCA-GTT-TTA-TGT-TAT-GCT-AAA-AAC-ATC-GTG-AGG, inserts a PstI site in the 3
-noncoding region. The
guaB coding sequence was amplified with Vent DNA polymerase
(New England Biolabs) using 30 cycles of the following protocol: 1-min
denaturation at 92 °C, 1-min annealing at 45 °C, 4-min extension
at 60 °C. The PCR reaction (100 µl) contained 200 µM
deoxynucleoside triphosphates, 4 mM MgSO4, 2 ng
of p68, 1.25 µM of each oligonucleotide, and 2 units of
polymerase. The PCR product was digested with NdeI and
PstI and ligated to the NdeI/PstI
fragment of pTactac (19). The resulting construct is designated pB9.
The guaB coding sequence of pB9 was completely sequenced
using a PRISM Dyedeoxy Terminator cycle sequencing kit (ABI) and an
Applied Biosystems 373A DNA sequencer at the Brandeis DNA Facility. No
unwanted mutations were introduced in the PCR reaction.
pB9 was
transformed into E. coli strain H712, which contains a
partial deletion of the E. coli guaB gene (20). An overnight culture of cells was diluted 200-fold into fresh LB broth containing 100 µg/ml ampicillin. After 1 h at 37 °C, 1 mM
IPTG was added to induce expression of IMPDH. The cells were harvested
after 13 h by centrifugation, resuspended in buffer A (50 mM Tris, pH 7.4, 1 mM dithiothreitol, 10%
glycerol), and frozen at 20 °C. All of the following manipulations
were performed at 4 °C. The cells were thawed and disrupted by
sonication. Debris was removed by centrifugation at 12,000 × g for 25 min. The supernatant was applied to a Cibacron blue
Sepharose column previously equilibrated in buffer A. IMPDH was eluted
in a linear gradient of 0 to 2 M KCl in buffer A. Fractions
containing IMPDH activity were pooled and diluted 4-fold with buffer A. IMP resin was added to the enzyme solution until no activity remained
in the supernatant. The resin was then poured into a column and washed
with buffer A. IMPDH was eluted with 1 mM IMP, 500 mM KCl in buffer A (IMPDH does not elute in the absence of
KCl). Table I summarizes the purification. Protein concentration was
measured using the Bio-Rad assay with IgG as a standard. Active sites
were titrated with
5-ethynyl-1-
-D-ribofuranosylimidazole-4-carboxamide 5
-monophosphate (9). N-terminal sequencing was performed by the Tufts
Medical School Protein Sequencing Facility. Electrospray ionization
mass spectroscopy was performed by the Harvard University Mass
Spectroscopy Laboratory.
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Sedimentation equilibrium experiments were performed on a Beckman model XL-A analytical ultracentrifuge monitoring absorbance at 280 nm. The rotor speed was 9000 rpm and the temperature was 20 °C. Samples contained 0.2-1.5 mg/ml enzyme, 50 mM Tris, pH 7.5, and 1 µM dithiothreitol in the presence and absence of 0.1 M KCl. Data were fit to the following equation, which describes the sedimentation of a single component system, using Origin Technical Graphics and Data Analysis (MicroCal):
![]() |
(Eq. 1) |
Assays were performed in 100 mM
KCl, 1 mM dithiothreitol, 50 mM Tris, pH 8.0. Activity was routinely assayed in the presence of 250 µM
IMP and 500 µM NAD at 25 °C. The production of XMP was monitored in crude extracts by absorbance at 290 nm ( = 5.4 mM
1 cm
1). The production of
NADH was monitored at 340 nm for purified enzyme assays (
= 6.2 mM
1 cm
1). Initial velocity data
were fit to the following equations using KinetAsyst and Kaleidagraph
software,
![]() |
(Eq. 2) |
![]() |
(Eq. 3) |
Competitive inhibition equation
![]() |
(Eq. 4) |
Uncompetitive inhibition equation
![]() |
(Eq. 5) |
PCR was
used to insert convenient restriction sites into the B. burgdorferi guaB gene, NdeI at the start site and
PstI in the 3-noncoding region, and the gene was cloned
into pTactac (19). The guaB gene was sequenced to ensure
that no mutations were introduced during PCR. This construct, pB9, was
transformed into E. coli strain H712, which lacks endogenous
IMPDH activity (20). H712 cells carrying pB9 could grow on minimal
medium, while cells carrying the parent plasmid, pTactac, cannot grow (data not shown). This result demonstrates that pB9 can complement the
IMPDH deficiency of H712 cells and indicates that pB9 expresses a
functional IMPDH.
Recombinant B. burgdorferi IMPDH was purified from H712
cells carrying pB9; expression was induced with 1 mM IPTG.
The purification was accomplished in two steps and high yield, using
Cibacron blue Sepharose and IMP affinity resins (Table
I). Purification of B. burgdorferi IMPDH closely resembled the purification of human type
II and E. coli IMPDHs (9, 21), with the exception that 0.5 M KCl is required in addition to 500 µM IMP
to elute the enzyme from the IMP resin. This observation suggests that
the B. burgdorferi enzyme may have a different requirement
for monovalent cations than other IMPDHs. The purified enzyme is >95%
homogeneous as judged by SDS-PAGE (Fig.
1).
Characterization of B. burgdorferi IMPDH
The N-terminal
sequence of purified IMPDH is Pro-Asn-Lys-Ile-Thr-Lys as determined by
Edman degradation. This sequence corresponds to the predicted
N-terminal sequence of B. burgdorferi IMPDH after removal of
the first Met. SDS-PAGE analysis of purified IMPDH shows a single band
at 44 kDa (Fig. 1), consistent with the molecular mass of 43,637 Da
calculated from the deduced amino acid sequence. This molecular mass
confirms that the IMPDH activity derives from the expression of
B. burgdorferi guaB because IMPDHs typically have a
molecular mass of ~55,000 Da (22). A single species with a molecular
mass of 43,660 Da is observed by electrospray ionization mass
spectroscopy (Fig. 2), which further
confirms the identity of B. burgdorferi IMPDH.
Fig. 3 shows equilibrium sedimentation
data for B. burgdorferi IMPDH. These data were fit to an
equation describing the sedimentation of a single ideal species with a
molecular mass of 43,660 Da (monomer), 87,320 Da (dimer), or 174,640 Da
(tetramer). Reasonable fits could not be obtained to monomeric and
dimeric species; the fit to a tetrameric species appears to be good
(Fig. 3). Nevertheless, the residuals of the tetrameric fit display a
nonrandom distribution, which can indicate an associating system. This
fit could not be improved by including terms for monomer-tetramer,
dimer-tetramer, or monomer-dimer-tetramer equilibria. The fit could be
improved slightly by including a tetramer-octamer equilibrium. Such
higher order aggregates have been observed in IMPDHs from other species (22, 23). No difference in sedimentation behavior was observed at
different enzyme concentrations (0.2 to 1.5 mg/ml) or in the presence
of 0.1 M KCl. These results indicate that B. burgdorferi IMPDH is a tetramer like other IMPDHs (22, 24, 25) and
may form higher order aggregates as also observed for other IMPDHs.
Steady State Kinetic Parameters
Initial velocity data were
collected at varying concentrations of IMP (7 to 980 µM)
and NAD (125 to 5000 µM). The initial velocity
versus IMP plots at fixed concentrations of NAD follow Michaelis-Menten kinetics (Fig.
4A). In contrast, substrate
inhibition is observed at high NAD concentrations (Fig. 4B).
Such substrate inhibition is commonly observed in IMPDHs and suggests
that product dissociation follows an ordered mechanism where NADH is
the first product released. Steady state parameters were derived by
first determining the apparent values of Vm for the
initial velocity versus IMP plots (as in Fig. 4A)
and replotting these values against NAD concentration. These data were
fit to Equation 3, which describes uncompetitive substrate inhibition:
kcat = 2.6 ± 0.3 s1,
Km (NAD) = 1100 ± 160 µM and
Kii (NAD) = 2300 ± 390 µM. The
value of Km (IMP) was derived by first determining
the apparent values of Vm for the initial velocity versus NAD plots using Equation 3 (as in Fig.
4B) and replotting these values against IMP concentration.
These data were fit to the Michaelis-Menten equation:
Km (IMP) = 29 ± 8 µM. These
values are comparable to those reported for other bacterial IMPDHs (22,
26). Thus B. burgdorferi guaB encodes an IMPDH with typical
kinetic properties. This observation demonstrates that the subdomain is
not required for IMPDH activity.
Monovalent Cation Dependence
B. burgdorferi IMPDH is inactive in the absence of K+ (<1% activity). High concentrations of K+ (>300 mM) inhibit the enzyme. As shown in Table II, NH4+ and K+ are interchangeable, while Cs+ is a much less effective activator. Neither Na+ or Li+ activate B. burgdorferi IMPDH; both of these cations are competitive inhibitors with respect to K+.
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While monovalent cation dependence is commonly observed among IMPDHs, the monovalent cation specificity varies significantly among IMPDHs. K+, NH4+, Na+, Tl+, and Rb+ activate human IMPDH, while Li+ has no effect on this enzyme (27). K+, but not NH4+, activates cowpea IMPDH (28), while Bacillus subtilis IMPDH will utilize K+, NH4+, Na+, Cs+, Li+, Tl+, and Rb+ (29). In contrast to these enzymes, Tritrichomonas foetus IMPDH does not require monovalent cations; this IMPDH retains 80% of its activity in the absence of K+ (8). These observations suggest that the monovalent cation binding site is a potential target for species specific inhibitors.
Inhibitor SensitivityXMP and GMP are competitive inhibitors with respect to IMP, Kis = 85 and 6 µM, respectively. The value of Kis for XMP is similar to those reported for other IMPDHs. In contrast, the Kis for GMP is approximately 15-fold lower than those usually observed for IMPDHs from both mammalian and bacterial sources (8, 30, 31).
Mycophenolic acid binds in the nicotinamide subsite of the dinucleotide site of IMPDH and prevents the hydrolysis of the covalent enzyme-XMP (E-XMP*) intermediate (17, 32, 33). Consistent with this mechanism of action, mycophenolic acid is an uncompetitive inhibitor of B. burgdorferi IMPDH with respect to both IMP and NAD (Table III). This observation suggests that B. burgdorferi IMPDH has an ordered mechanism of product release where NADH dissociates before hydrolysis of E-XMP*, as observed with other IMPDHs (34). The Kii for mycophenolic acid inhibition of B. burgdorferi IMPDH is 103-fold greater than that for mammalian IMPDHs, as is typical of microbial IMPDHs (8, 7, 35). These results suggest that the dinucleotide site of microbial IMPDHs is a target for species-specific inhibitors.
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CH2-TAD is a nonhydrolyzable analog of the active metabolite of the anti-tumor drug tiazofurin. CH2-TAD is also an uncompetitive inhibitor of B. burgdorferi IMPDH with respect to both IMP and NAD. This observation suggests that CH2-TAD also binds to E-XMP*. The Kii of CH2-TAD = 1.0 µM is approximately 20-fold greater than that of mammalian IMPDHs (36), further demonstrating a difference in the dinucleotide sites of microbial and mammalian IMPDHs.
SummaryWe have demonstrated that the guaB homolog of B. burgdorferi encodes IMPDH. This result demonstrates that the subdomain is not required for IMPDH activity. The function of this subdomain is unknown. In addition, we show that the monovalent cation and dinucleotide binding sites of B. burgdorferi IMPDH differ significantly from mammalian IMPDHs. Therefore these sites are targets for the design of species-specific inhibitors of IMPDH. Such inhibitors could be used to treat Lyme disease.
This manuscript is Publication No. 1813 from the Department of Biochemistry, Brandeis University.