(Received for publication, September 16, 1996, and in revised form, October 30, 1996)
From the Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254
Mycophenolic acid (MPA) is a potent and specific
inhibitor of mammalian inosine-monophosphate dehydrogenases (IMPDH);
most microbial IMPDHs are not sensitive to MPA. MPA-resistant mutants of human IMPDH type II were isolated in order to identify the structural features that determine the species selectivity of MPA.
Three mutant IMPDHs were identified with decreased affinity for MPA.
The mutation of Gln277 Arg causes a 9-fold increase in
the Ki of MPA, a 5-6-fold increase in the
Km values for IMP and NAD, and a 3-fold decrease in
kcat relative to wild type. The mutation of
Ala462
Thr causes a 3-fold increase in the
Ki for MPA, a 2.5-fold increase in the
Km for NAD, and a 1.5-fold increase in
kcat. The combination of these two mutations
does not increase the Ki for MPA, but does increase
the Km for NAD 3-fold relative to Q277R and
restores kcat to wild type levels. Q277R/A462T
is the first human IMPDH mutant with increased Ki for MPA and wild type activity. The third mutant IMPDH contains two
mutations, Phe465
Ser and Asp470
Gly.
Ki for MPA is increased 3-fold in this mutant enzyme, and Km for IMP is also increased 3-fold,
while the Km for NAD and
kcat are unchanged. Thus increases in the
Ki for MPA do not correlate with changes in
Km for either IMP or NAD, nor to changes in
kcat. All four of these mutations are in
regions of the IMPDH that differ in mammalian and microbial enzymes,
and thus can be structural determinants of MPA selectivity.
Inosine-monophosphate dehydrogenase
(IMPDH)1 catalyzes the oxidation of IMP to
XMP with the concomitant conversion of NAD to NADH (Fig. 1). This
reaction is the rate-limiting step in guanine nucleotide biosynthesis,
and rapidly growing cells have increased levels of IMPDH (1).
Inhibitors of IMPDH have antiproliferative activity and are used
clinically for cancer, viral, and immunosuppressive chemotherapy
(2, 3, 4). Moreover, differences in the properties of microbial and
mammalian IMPDHs suggest that species-selective IMPDH inhibitors can be
designed, which will be useful for anti-infective chemotherapy (5, 6, 7).
Two human IMPDH isozymes exist; type I is constitutively expressed,
while type II is expressed in rapidly proliferating cells (8, 9, 10, 11). The
IMPDH reaction involves attack of Cys331 (human type II
numbering) at the 2-position of IMP, followed by expulsion of the
hydride to NAD (Fig. 1). The resulting covalent E-XMP* intermediate is subsequently hydrolyzed to XMP (12,
13).
Mycophenolic acid (MPA) is a potent and specific inhibitor of mammalian
IMPDHs, and a MPA derivative, mycophenolate mofetil, is a promising
immunosuppressive drug (14, 15). MPA affinity varies greatly among
microbial and mammalian IMPDH, for example Ki = 22 nM for human IMPDH, 500 nM for the
Bacillus subtilis, 20 µM for the
Escherichia coli and 14 µM for the
Tritrichomonas foetus enzymes
(16, 17, 18).2 MPA traps the E-XMP
intermediate in mammalian IMPDHs (Fig. 1), and the crystal structure of
the E-XMP·MPA complex of IMPDH from Chinese hamster has
recently been solved (12, 19, 20). MPA stacks against E-XMP
in the likely nicotinamide binding site, as predicted by multiple
inhibitor studies (17). Of the residues that contact MPA, only
Arg322 and Gln441 differ in mammalian and
microbial IMPDHs; Arg322 is replaced by Lys, and
Gln441 is replaced by Glu (21, 22, 23). While the mutation of Gln441 Ala decreases MPA sensitivity by 20-fold,
activity is also decreased 20-fold (20). Mutations at
Arg322 have not been reported. Moreover, both B. subtilis and E. coli IMPDHs contain Lys322
and Glu441, although the Ki values of
MPA for these enzymes vary by 40-fold. Thus residues 322 and 441 cannot
be the only structural determinants of MPA selectivity.
Random mutagenesis followed by selection for the ability to grow in the
presence of MPA can identify mutations in IMPDH that confer MPA
resistance. Selection for MPA resistance has previously been reported
in both mammalian and parasite systems. In most cases MPA resistance
resulted from increased expression of IMPDH, usually via gene
amplification (24, 25, 26). Mutant IMPDHs with altered sensitivity to MPA
have been reported in murine lymphoma and leukemia cells, although
identity of these mutations and their effect on enzyme activity were
not characterized (27, 28). A MPA-resistant neuroblastoma cell line has
been isolated in which a mutant IMPDH is overexpressed by gene
amplification (29). While this mutant IMPDH is less sensitive to MPA,
it is also much less active than wild type. This MPA-resistant IMPDH
contains two mutations: Thr333 Ile and
Ser351
Tyr. Recent mutagenesis experiments suggest that
resistance results from the alteration of Thr333 (20). This
residue is strictly conserved in all IMPDHs sequenced to date, and
therefore cannot be a determinant of species selectivity. In addition
to these examples, MPA resistance can also result from alterations in
purine salvage pathways or in enzymes that utilize guanine nucleotides
and from inactivation of MPA via glucuronidation (30, 31, 32).
We have isolated and characterized MPA-resistant mutants of human type II IMPDH in order to explore the structural basis of the species selectivity of MPA. We have identified three mutant enzymes that have a decreased sensitivity to MPA. These enzymes are 3-8-fold less sensitive to MPA. Four substitutions are identified in regions of the protein that are conserved in mammalian IMPDHs, but different in microbial enzymes. Thus these residues may be structural determinants of MPA sensitivity. Interestingly, these substitutions are in residues that do not contact MPA.
IMP, NAD, and mycophenolic acid were purchased from Sigma. Plasmid pHIMP containing human IMPDH type II cDNA was the generous gift of Dr. Frank Collart (8).
Construction of an Expression System for Human IMPDH Type IIThe 1.5-kb NcoI/SalI fragment containing
the IMPDH coding sequences from a partial digest of pHIMP was ligated
to the NcoI/SalI fragment of the pBluescript II
KS+-based vector pST (33) (pBluescript II KS+
from Stratagene). This construct (pSI) was used as a template for
Kunkel mutagenesis (34). Four silent mutations were inserted into the
IMPDH coding sequences, creating four unique restriction sites (pSI5).
The following oligonucleotides were used (mismatched bases are
underlined): NdeI at position 1, AAC AAA CAC C
ATG GCC GAC TAC; XhoI at position 473, ATC ATC TC
C AGG GAC ATT GAT; KpnI at position 750, GCC ATT
GG
AC
CAT GAG GAT GAC; SacII at
position 1232, AAG AAA TA
CGC GGT ATG GGT.
This construct was sequenced in its entirety to confirm that only the desired mutations were introduced. One difference from the published cDNA sequence was noted (8); we found that the sequence of bases 608-611 is GCA GGC, not CGC AGC as reported previously. As a consequence, residues 190 and 191 are Ala and Gly, respectively, rather than Arg and Ser. This sequence was also found in pHIMP, indicating that this discrepancy did not arise during the construction and mutagenesis of pSI. Our results agree with the cDNA sequence reported by Natsumeda in this region of human IMPDH type II (9) and with sequences of the IMPDH type II gene (35, 36).
The 1.5-kb NdeI (filled in by Klenow
extension)/HindIII fragment of pSI5 containing the IMPDH
coding sequences was ligated to the NcoI (treated with S1
nuclease to create blunt ends)/HindIII fragment of pKK233-2
to create pHIA5 (37). This construct can complement the guaB
deficiency of H712 cells. The junction of the
NcoI-NdeI sites contains the sequence (ribosome
binding site is underlined) AACAGAC-A(1)TG, which
indicates that a T normally found at
1 was deleted during subcloning.
Similar expression systems for human type II IMPDH have been reported
previously (18, 38, 39).
A library of randomly mutagenized pHIA5 was created by transforming pHIA5 into the mutD5 E. coli strain NR9072 (40). The plasmid was reisolated from a culture grown on LB broth containing 100 µg/ml ampicillin. This mutagenized plasmid was used to transform E. coli strain H712, which carries a partial deletion in the guaB gene (41).3 MPA-resistant colonies were selected by growth on minimal media containing 200 µM mycophenolic acid, 9.6 µg/ml tryptophan, 48 µg/ml histidine, 5.0 µg/ml tyrosine, 0.1 µg/ml thymine, 2% glucose, and 25 µg/ml ampicillin. Plasmid was isolated from these colonies and used to transform H712 cells. The new transformants were tested for the ability to grow on minimal medium in the presence of MPA as described above.
Construction of L30F, Q277R, and Q277R/A462TThe plasmid pHIMA17, which expresses L30F/Q277R IMPDH, was digested with XhoI and HindIII. The 1.2-kb fragment containing the L30F mutation was ligated to the 5.0-kb XhoI/HindIII fragment of pHIA5 to produce pHIMA51, which expresses L30F IMPDH. The 5.0-kb fragment of pHIMA17 was ligated to the 1.2-kb fragment of pHIA5 to produce pHIMA52, which expresses Q277R IMPDH. Similarly, the 0.7-kb XhoI/SacII fragment of pHIMA18 containing the A462T mutation was ligated to the 5.5-kb XhoI/SacII fragment of pHIMA17 to produce pHIMA53, which expresses Q277R/A462T IMPDH.
DNA SequencingDNA sequencing was performed using either 35S-dATP with a Sequenase kit (U. S. Biochemical Corp.) or a PRISM Dyedeoxy Terminator Cycle Sequencing kit (Applied Biosystems, Inc.) and an Applied Biosystems 373A DNA sequencer at the Brandeis Sequencing Facility.
IMPDH PurificationWild type enzyme was purified in two steps using affinity chromatography. H712 cells carrying pHIA5 were grown overnight in LB broth containing 1 mM IPTG and 100 µg/ml ampicillin. The cells were harvested by centrifugation and resuspended in 50 mM Tris, pH 7.5, 1 mM dithiothreitol, 10% glycerol (Buffer A). The cells were disrupted by sonication and cell debris removed by centrifugation at 12,000 × g for 20 min. The crude lysate was applied to a Cibacron Blue Sepharose column pre-equilibrated in Buffer A. IMPDH was eluted in a linear gradient of 0-1 M KCl in Buffer A. The IMPDH containing fractions were pooled, diluted 4-fold in Buffer A, and applied to an IMP affinity column (42). IMPDH was eluted in Buffer A containing 0.5 mM IMP. The preparation is homogeneous as judged by SDS-polyacrylamide gel electrophoresis analysis. IMPDH was concentrated using Centriprep filters (Amicon). IMP was removed by dialysis against buffer A as needed. Protein was quantified using the Bio-Rad assay with IgG as a standard. This assay overestimates the IMPDH concentration by a factor of 2.6 (7), and measurements were adjusted accordingly. The concentration of IMPDH determined by this method agreed with that determined in the MPA inhibition experiments.
Purification of Mutant IMPDHsF456S/D470G, D255G, M482I and G340E were purified as described above. A462T did not bind to the IMP affinity column. Therefore crude lysate of A462T was chromatographed on a Bio-Gel A5m column. A462T containing fractions were applied to a Cibacron Blue Sepharose column and eluted as above. This preparation was homogeneous as determined by SDS-polyacrylamide gel electrophoresis. L30F/Q277R, L30F, and Q277R are unstable in the absence of IMP. Therefore fractions from the Cibacron Blue Sepharose column were collected in tubes containing IMP such that the final IMP concentration was 0.5 mM. Activity was lost upon further purification of these enzymes. No NADH oxidase or phosphatase activity was detected in these preparations, which suggests that the partially purified enzymes are suitable for kinetic studies. It should be noted that the specific activity of wild type IMPDH does not increase after the Blue Sepharose column, which indicates that this single purification step yields > 80% homogeneous enzyme preparations. The concentrations of L30F/Q277R, L30F, and Q277R were determined in the MPA inhibition experiment. Q277R/A462T is also unstable in the absence of IMP. The Cibacron Blue Sepharose fractions of Q277R/A462T were further purified on a POROS CM cation exchange column using a BioCAD Sprint perfusion chromatography system (PerSeptive Biosystems). The column was equilibrated in 7 mM Hepes, 7 mM Mes, 7 mM acetate buffer, pH 6.0 (Buffer B). Enzyme was eluted in a linear gradient of 0-1 M NaCl in Buffer B into test tubes containing IMP.
Enzyme AssaysThe standard assay solution contains 50 mM Tris, pH 8.0, 100 mM KCl, 1 mM dithiothreitol, 3 mM EDTA, 125 µM IMP, and 100 µM NAD. The production of NADH is monitored by the change in absorbance at 340 nm on a Hitachi U-2000 spectrophotometer. For Km determinations, IMP and NAD are varied as appropriate. Enzymes were dialyzed to remove IMP prior to Km determinations. MPA affinity was determined by varying NAD and MPA concentration in the presence of 1 mM IMP. All assays were performed at 25 °C.
Data AnalysisMichaelis-Menten parameters were determined by fitting initial rate data to a sequential mechanism (Equation 1) using KinetAsyst software.
![]() |
(Eq. 1) |
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(Eq. 2) |
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The cDNA
(1.5 kb) of human IMPDH type II was modified by silent mutations to
insert restriction sites at the following bases: 1 (NdeI),
473 (XhoI), 750 (KpnI), and 1232 (SacII). The modified cDNA was cloned into the E. coli expression vector pKK233-2 as described under "Materials
and Methods" (37). The NdeI site was lost in the cloning
process. This construct, pHIA5, expresses human IMPDH type II under the
control of the trc promoter. pHIA5 was transformed into
E. coli strain H712, which contains a partial deletion in
guaB, the gene encoding IMPDH (41). H712 cells carrying pHIA5 can grow on minimal medium in the absence of guanine, which indicates that IMPDH is expressed in the absence of IPTG. These cells
cannot grow on minimal medium in the presence of 200 µM MPA (data not shown).
A library of randomly mutagenized IMPDH was generated using a mutD5 strain of E. coli. This library was transformed into E. coli strain H712. MPA-resistant clones were selected by growth on minimal medium containing 200 µM MPA. Thirty clones were selected from 106 colonies screened in two separate experiments. Plasmids were isolated from these clones and used to transform H712 cells. All of the retransformed clones could grow on minimal medium in presence of 200 µM MPA, which indicates that MPA resistance is contained on the plasmid. Twenty-three of these clones were chosen for further characterization.
Screen of IMPDH Activity and MPA ResistanceMPA resistance can result either from a decrease in the affinity of IMPDH for MPA or from an increase in IMPDH levels. Therefore, crude extracts were prepared from the MPA-resistant clones in order to screen for clones that might contain altered IMPDHs. Crude extracts were prepared from cultures grown in LB broth in the presence of IPTG. These growth conditions were chosen in order to maximize the amount of IMPDH in the crude extracts. IMPDH activity was assayed in the presence of 125 µM IMP and 200 µM NAD. These substrate concentrations are saturating for wild type IMPDH. The relative specific activities of these preparations are shown in Table I. Six of the crude extracts had relative specific activities within 0.5-1.5-fold of wild type, 14 had specific activities greater than 1.5-fold, and three had specific activities less than 0.5-fold of wild type. The IC50 for MPA inhibition was also measured under these conditions (Table I). Six clones were identified with relative IC50 values of 4 or more: MA5, MA9, MA14, MA17, MA18, and MPA11. All of these mutants have specific activities less than 1.6-fold of wild type, which suggests that MPA resistance may result from alteration of IMPDH rather than an increase in IMPDH concentrations.
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Plasmids isolated from the
MPA-resistant colonies were sequenced in order to identify mutations in
IMPDH. Of the six clones identified above, four express mutant IMPDHs.
MA5 and MA18 express an IMPDH where Ala462 is changed to
Thr (henceforth this mutant IMPDH is denoted A462T). MA17 expresses an
IMPDH with two mutations; Leu30 is changed to Phe and
Gln277 is changed to Arg (L30F/Q277R). MPA11 also expresses
an IMPDH with two mutations; Phe456 is changed to Ser and
Asp470 is changed to Gly (F456S/D270G). A mutation is found
at 6 in the IMPDH coding sequence of MA14. This mutation could confer MPA resistance by increasing the expression of IMPDH. No mutation is
present in the IMPDH coding sequences in MA14, which suggests that a
mutation in another region of the plasmid must be responsible for MPA
resistance.
The remaining 17 clones were also sequenced. Overall, 17 mutations were
identified in the IMPDH coding and adjacent sequences of the 23 plasmids (Tables I and II). Only two of the mutations were transversions, which is consistent with previous observations of
mutD5-mediated mutagenesis in rich media (40). Five clones contained substitutions at 6, which could increase expression of
IMPDH and thus cause MPA resistance. A total of five clones were
isolated containing the mutation of Ala462
Gly. The MPA
IC50 values of these clones varied from 2- to 15-fold of
wild type. This variability could result from the presence of mutations
elsewhere on the plasmid. Alternatively, this variability might result
from the difficulties inherent in working with crude extracts. In
addition to the mutations described above, one clone was isolated with
each of the following single mutations: Met482
Ile,
Gly340
Glu, and Asp255
Gly. No
mutations could be identified in the IMPDH coding sequences of the
remaining eight clones.
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The mutant proteins were purified as described under "Materials and Methods." A462T did not bind to an IMP affinity column, which indicates a change in IMP binding. L30F/Q277R is unstable in the absence of IMP. The remaining mutant IMPDHs have purification characteristics similar to wild type.
The Ki values for MPA inhibition of wild type and mutant IMPDHs are summarized in Table III. MPA is a tight binding inhibitor of IMPDH under the conditions in these assays. In all cases, the data best fit an uncompetitive inhibition mechanism versus NAD. However, while a competitive mechanism can be definitively eliminated, the fit to a noncompetitive mechanism is only marginally worse than the uncompetitive fit. The parameters for human IMPDH type II are similar to those reported elsewhere (18, 39). The Ki for MPA inhibition is increased relative to wild type in the three mutant enzymes originally identified in the crude extract screen. The Ki of MPA is increased 3-fold in A462T. This mutation was isolated five times; although these isolations do not necessarily represent independent mutational events, this observation suggests that MPA resistance can result from this modest decrease in MPA affinity. A 3-fold increase in Ki is also observed for F456S/D470S. The Ki of MPA is increased 8-fold in L30F/Q277R. No increase in the Ki for MPA is observed in the other mutant IMPDHs, in agreement with the crude extract experiments.
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Alterations are also observed in the steady state kinetic parameters of the MPA-resistant mutants (Table III). In A462T, Km for IMP is increased by 2.5-fold relative to wild type, and kcat is increased 1.5-fold, while no significant change is observed in the Km for NAD. In L30F/Q277R, Km for NAD is increased by 4.5-fold relative to wild type, and kcat is decreased 5.5-fold. Unfortunately, the Km for IMP could not be measured because this enzyme is unstable in the absence of IMP. In F456S/D470S, neither the Km for IMP nor the Km for NAD are significantly changed, although kcat is decreased 2.4-fold. The Km values and kcat values of D255G, M482I, and G340E are indistinguishable from wild type.
Characterization of Single Mutants L30F and Q277RL30F/Q277R
is the only mutant with a dramatic increase in the
Ki of MPA. Therefore the single mutants L30F and
Q277R were constructed, purified, and characterized in order to
determine if both mutations are necessary for MPA resistance. The
Ki for MPA inhibition of L30F is similar to wild
type, while the Ki for MPA inhibition of Q277R is
6-fold greater than wild type. Thus the Gln277 Arg
mutation is sufficient to confer MPA resistance. The
Km for NAD of L30F is also similar to wild type,
although kcat is decreased by 5-fold. The
Km values of both IMP and NAD are increased
5-6-fold in Q277R, while kcat is decreased by
3-fold.
A double mutant was
constructed in order to determine if the two mutations that confer MPA
resistance, Gln277 Arg and Ala462
Thr,
function independently. The Ki for MPA inhibition of
Q277R/A462T is similar to Q277R; thus the effects of the mutations are
not additive and the mutations are not independent. Interestingly, the
Km for IMP of Q277R/A462T is similar to Q277R;
however, the Km for NAD is increased 3-fold and
kcat is increased 4-fold. Thus the
Ala462
Thr mutation restores the defect in
kcat caused by the Gln277
Arg
mutation. Q277R/A462T is the first mutant IMPDH with wild type levels
of activity and decreased sensitivity to MPA.
We have
used a random mutagenesis and selection approach to identify mutations
in human IMPDH type II that can confer MPA resistance. Human IMPDH type
II was expressed in an E. coli strain containing a deletion
in guaB. This strain cannot grow in the presence of MPA and
was used to select MPA-resistant clones. Unlike previous approaches,
this method can screen large numbers of clones and other mutations in
the purine biosynthetic pathways can be readily eliminated. Twelve
MPA-resistant clones were identified, which contained mutations in
IMPDH. Three mutant IMPDHs were isolated with decreased
Ki values for MPA: L30F/Q277R, A462T, and
F456S/D470G. The greatest increase in the Ki for MPA
(8-fold) is observed in L30F/Q277R. This increase can be attributed to
the substitution of Arg for Gln277. A 3-fold increase in
the Ki for MPA is observed in A462T. A 3-fold
increase in MPA Ki is also observed in F456S/D470G.
Since this increase is modest, no attempt has been made to determine if
both mutations are required for the change in MPA affinity. Three
additional mutant IMPDHs were selected in the screen for MPA
resistance: D255G, M482I, and G340E. These mutations had no measurable
effect on the Ki of MPA, or on the steady state
kinetic parameters. It is possible that these mutations are fortuitous
(like the Leu30 Phe mutation) and that additional
mutations, present elsewhere on the plasmid, confer MPA resistance.
Alternatively, the in vitro assay conditions may not mimic
in vivo conditions closely enough to reveal differences in
enzyme function.
MPA binds in the nicotinamide site and traps the E-XMP* intermediate (12, 20). Therefore MPA resistance can result from a change in the MPA/nicotinamide site and might be manifest in the Km for NAD. Since MPA binding involves a stacking interaction with E-XMP*, changes in the IMP site can also increase the Ki for MPA. In addition, the Ki for MPA will be increased if E-XMP* no longer accumulates during the IMPDH reaction. For example, a mutation that changed the rate of a conformational change could change the accumulation of E-XMP*. These mechanisms need not be mutually exclusive; indeed, while the Km values of both NAD and IMP are higher in IMPDH from T. foetus and E. coli than in the human enzyme, the kcat values are also higher in the microbial enzymes, which suggests that both mechanisms are important. A462T, Q277R, and Q277R/A462T display increases in both the Km values for IMP and the Km values for NAD, while the substrate Km values of F456S/D270G are similar to wild type. The kcat for A462T is 1.5-fold higher than wild type, and Q277R/A462T is similar to wild type, while the other mutations decrease kcat. These results suggest that the mutations may both change the MPA binding site and the accumulation of E-XMP*.
Structural Context of Mutations That Confer MPA ResistanceUnfortunately, the coordinates of the structure of the
E-XMP·MPA complex are not yet available (20), so it is
difficult to evaluate the effects of these mutations on the structure
of IMPDH. In addition, while the residues that contact MPA and the E-XMP intermediate appear to be clearly delineated, the
residues that contact NAD have yet to be identified. These residues are also expected to influence MPA sensitivity. IMPDH is an /
barrel; the active site is located in loops on the C-terminal ends of the
strands. The active site Cys331 is found in the loop
between
6 and
6 (residues 325-342), and additional active site
residues are in the loops between
4 and
4 (residues 275-280) and
8 and
8 (residues 400-450). The large loop after
8 forms a
flap over the active site; the flap residues interact with both
E-XMP and MPA.
Gln277 is in the loop between 4 and
4. The adjacent
residues, Asp274, Ser275, and
Ser276, contact MPA in the structure. Thus, while
Gln277 does not contact MPA directly, substitutions at
position 277 can easily affect the residues that do contact MPA. While
residues 274-276 are conserved among IMPDHs, 277 is His and Asp in the IMPDHs from E. coli and T. foetus, respectively
(21, 23). Thus residue 277 may be a determinant of species selectivity. In addition, residues 279-281 are involved in intersubunit contacts in
the IMPDH tetramer. Therefore, substitutions at 277 may also affect the
function of the adjacent subunit. Ala462 is located in the
middle of
8 and would appear to be removed from the active site.
Phe456 is also located in
8, at the beginning of the
helix. Interestingly, residue 462 is the n + 6 residue from
456, and thus would be on the same side of the helix.
Asp470 is the first residue in the loop at the end of
8,
and would therefore be on the opposite end of the barrel from the
active site, although on the same side of the helix as residues 456 and 462. This cluster of mutations suggests an important role for
8 in
the IMPDH reaction. It is possible that
8 may influence the flap,
and thus modulate the accumulation of E-XMP* and MPA affinity. Helix
8 is highly conserved in mammalian IMPDH, but varies
widely in IMPDHs from other sources (Fig. 2). Therefore,
8 may be a structural determinant of MPA selectivity.
We thank Catherine Spicer for the initial characterization of D255G, Rebecca Meyers for assistance with the DNA sequencing, and Kathleen Kerr and Annette Pasternak for comments on the manuscript.