Isolation and Characterization of Mycophenolic Acid-resistant Mutants of Inosine-5'-monophosphate Dehydrogenase*

(Received for publication, September 16, 1996, and in revised form, October 30, 1996)

Thalia Farazi Dagger , Joshua Leichman §, Thanawath Harris , Marguerite Cahoon and Lizbeth Hedstrom par

From the Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

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 right-arrow 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 right-arrow 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 right-arrow Ser and Asp470 right-arrow 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.


INTRODUCTION

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).


Fig. 1. The IMPDH reaction.
[View Larger Version of this Image (9K GIF file)]


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 right-arrow 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 right-arrow Ile and Ser351 right-arrow 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.


MATERIALS AND METHODS

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 II

The 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<UNL>AT</UNL> ATG GCC GAC TAC; XhoI at position 473, ATC ATC TC<UNL>G:AG</UNL>C AGG GAC ATT GAT; KpnI at position 750, GCC ATT GG<UNL>T</UNL> AC<UNL>C</UNL> CAT GAG GAT GAC; SacII at position 1232, AAG AAA TA<UNL>C</UNL> 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) <UNL>AGGA</UNL>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).

Isolation of MPA-resistant Clones

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/A462T

The 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 Sequencing

DNA 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 Purification

Wild 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 IMPDHs

F456S/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 Assays

The 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 Analysis

Michaelis-Menten parameters were determined by fitting initial rate data to a sequential mechanism (Equation 1) using KinetAsyst software.
v=V<SUB>m</SUB>AB/(K<SUB>ia</SUB>K<SUB>b</SUB>+K<SUB>a</SUB>B+K<SUB>b</SUB>A+AB) (Eq. 1)
v is the initial velocity, Vm is the maximal velocity, A is the concentration of IMP, B is the concentration of NAD, Kia is the dissociation constant for IMP from the binary EA complex, Kb is the Km for NAD, and Ka is the Km for IMP. MPA affinity was determined by fitting initial rate data to Equation 2, which describes a tight binding uncompetitive inhibitor (43, 44).
v=(v<SUB>0</SUB>/2E){[E−<UP>I</UP>−K<SUB>i</SUB>(1+K<SUB>b</SUB>/B)]+ (Eq. 2)
([<UP>I</UP>+K<SUB><UP>i</UP></SUB>(1+K<SUB>b</SUB>/B)+E]<SUP>2</SUP>+4K<SUB>i</SUB>[1+K<SUB>b</SUB>/B]E)<SUP>0.5</SUP>}
I is the concentration of MPA, v0 is the initial velocity in the absence of I, E is the concentration of IMPDH active sites, and Ki is dissociation constant for MPA.


RESULTS

Expression of Human IMPDH Type II in E. coli

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).

Isolation of MPA-resistant Clones

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 Resistance

MPA 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.

Table I.

Screen of IMPDH activity and IC50 for MPA inhibition in crude extracts prepared from MPA-resistant clones

Crude extracts were prepared from LB cultures containing 100 µg/ml ampicillin and 1 mM IPTG. Assays contained 125 µM IMP, 200 µM NAD in 100 mM KCl, 1 mM dithiothreitol, 50 mM Tris, pH 8.0, at 25 °C. The specific activity of the wild type IMPDH crude extract is 0.015 µmol min-1 mg-1. "MA" and "MPA" denote clones isolated in different experiments.
Clone Specific activity (relative) IC50 (relative) Mutation

Wild type 1.0 1.0 None
MA1 1.5 1.2 None
MA2 1.5 1.0  -6A right-arrow C
MA3 2.0 2.0 Ala462 right-arrow Thr
MA4 2.7 1.2 Asp255 right-arrow Gly
MA5 1.6 10 Ala462 right-arrow Thr
MA6 2.6 2.0 Ala462 right-arrow Thr
MA7 2.1 1.4 None
MA8 1.1 1.5 Met482 right-arrow Ile
MA9 1.1 15 None
MA10 2.3 1.4  -6A right-arrow G
MA11 1.7 1.0 None
MA12 1.8 2.0 Ala462 right-arrow Thr
MA13 2.9 1.6 None
MA14 1.5 4.0  -6A right-arrow G
MA15 0.3 0.4  -6A right-arrow C
MA16 2.3 3  -6A right-arrow G
MA17 0.2 4 Leu30 right-arrow Phe/Gln277 right-arrow Arg
MA18 1.9 15 Ala462 right-arrow Thr
MPA4 1.7 0.9 None
MPA11 0.4 >4 Phe456 right-arrow Ser/Asp470 right-arrow Gly
MPA12 1.7 1.1 None
MPA13 1.0 0.8 Gly340 right-arrow Glu
MPA15 2.1 1.1 None

Sequencing of MPA-resistant IMPDHs

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 right-arrow 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 right-arrow Ile, Gly340 right-arrow Glu, and Asp255 right-arrow Gly. No mutations could be identified in the IMPDH coding sequences of the remaining eight clones.

Table II.

Mutations contained in the IMPDH coding sequence of MPA-resistant mutants

A library of randomly mutated pHIA5, which expressed human IMPDH type 2, was obtained by isolating the plasmid from a mutD5 strain of E. coli. The library was transformed into strain H712, which contains a partial deletion of the E. coli guaB gene. Clones were selected for the ability to grow in the presence of 200 µM MPA. Plasmids were isolated from the resistant clones and used to transform H712. The new transformants were screened for the ability to grow in the presence of 200 µM MPA. The IMPDH cDNAs of the plasmids surviving the second screen were sequenced to identify mutations that might confer MPA resistance.
Base position Mutation Substitution No. of clones isolated

 -6 right-arrow G 3
3 right-arrow C 2
88a right-arrow T Leu30  right-arrow Phe 1
765 right-arrow G Asp255  right-arrow Gly 1
830a right-arrow G Gln277  right-arrow Arg 1
1019 right-arrow A Gly340  right-arrow Glu 1
1367b right-arrow C Phe456  right-arrow Ser 1
1384 right-arrow A Ala462  right-arrow Thr 5
1409b right-arrow G Asp470  right-arrow Gly 1
1446 right-arrow A Met482  right-arrow Ile 1
No mutations 8

a  One clone contained both mutations.
b  One clone contained both mutations.

Characterization of Mutant IMPDHs

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.

Table III.

Ki for MPA inhibition and steady state kinetic parameters of wild type and mutant IMPDHs

Conditions are as described in Materials and Methods.
Enzyme Ki MPA Km IMP Km NAD kcat

nM µM µM s-1
Wild type 22  ± 8 13  ± 1 21  ± 5 0.41  ± 0.05
A462T 62  ± 12 33  ± 2 37  ± 7 0.61  ± 0.1
L30F/Q277R 180  ± 60 ND 180  ± 13 0.10  ± 0.02
F456S/D470G 71  ± 19 8  ± 1 17  ± 1 0.17  ± 0.05
D255G 17  ± 7 16.8  ± 0.6 10  ± 2 0.35  ± 0.02
M482I 6  ± 3 23  ± 4 10  ± 2 0.40  ± 0.02
G340E 11  ± 4 7  ± 3 12  ± 2 0.55  ± 0.04
L30F 19  ± 3 ND 15  ± 1 0.075  ± 0.015
Q277R 130  ± 20 80  ± 17 105  ± 17 0.13  ± 0.06
Q277R/A462T 140  ± 20 85  ± 4 320  ± 30 0.51  ± 0.16

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 Q277R

L30F/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 right-arrow 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.

Characterization of Q277R/A462T

A double mutant was constructed in order to determine if the two mutations that confer MPA resistance, Gln277 right-arrow Arg and Ala462 right-arrow 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 right-arrow Thr mutation restores the defect in kcat caused by the Gln277 right-arrow Arg mutation. Q277R/A462T is the first mutant IMPDH with wild type levels of activity and decreased sensitivity to MPA.


DISCUSSION

Isolation of MPA-resistant Mutants of Human IMPDH

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 right-arrow 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.

Effects of the MPA-resistant Mutations on the Function of IMPDH

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 Resistance

Unfortunately, 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 alpha /beta barrel; the active site is located in loops on the C-terminal ends of the beta  strands. The active site Cys331 is found in the loop between beta 6 and alpha 6 (residues 325-342), and additional active site residues are in the loops between beta 4 and alpha 4 (residues 275-280) and beta 8 and alpha 8 (residues 400-450). The large loop after beta 8 forms a flap over the active site; the flap residues interact with both E-XMP and MPA.

Gln277 is in the loop between beta 4 and alpha 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 alpha 8 and would appear to be removed from the active site. Phe456 is also located in alpha 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 alpha 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 alpha 8 in the IMPDH reaction. It is possible that alpha 8 may influence the flap, and thus modulate the accumulation of E-XMP* and MPA affinity. Helix alpha 8 is highly conserved in mammalian IMPDH, but varies widely in IMPDHs from other sources (Fig. 2). Therefore, alpha 8 may be a structural determinant of MPA selectivity.


Fig. 2. Sequence alignment and helix alpha 8. Sequences were aligned using PILEUP in the GCG software package. References are as follows: human IMPDH type II and Chinese hamster IMPDH type II (8), E. coli (21), B. subtilis (22), and T. foetus (23).
[View Larger Version of this Image (18K GIF file)]



FOOTNOTES

*   This work was supported in part by a Howard Hughes undergraduate research summer fellowship (to T. F.), Nathan and Bertha Richter undergraduate research fellowships (to J. L. and T. H.), a grant from the Massachusetts Division of the American Cancer Society (to L. H.), and a grant from the Lucille P. Markey Charitable Trust (to Brandeis University). This is Contribution 1807 from the Graduate Department of Biochemistry, Brandeis University. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Present address: Dept. of Molecular Biology & Pharmacology, Washington University, St. Louis, MO 63105.
§   Present address: Dept. of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-1569.
   Present address: Dept. of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107.
par    Searle Scholar and a Beckman Young Investigator. To whom correspondence should be addressed: Graduate Dept. of Biochemistry, MS 009, Brandeis University, 415 South St., Waltham, MA 02254. Tel.: 617-736-2333; Fax: 617-736-2349.
1    The abbreviations used are: IMPDH, inosine-monophosphate dehydrogenase; MPA, mycophenolic acid; IPTG, isopropyl-1-thio-beta -D-galactopyranoside; kb, kilobase pair(s); Mes, 4-morpholineethanesulfonic acid.
2    N. Benfield and L. Hedstrom, unpublished observations.
3    K. Kerr and L. Hedstrom, unpublished observations.

Acknowledgments

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.


REFERENCES

  1. Weber, G. (1983) Cancer Res. 43, 3466-3492 [Medline] [Order article via Infotrieve]
  2. Robins, R. (1982) Nucleosides & Nucleotides 1, 35-44
  3. Allison, A. C., Kowalski, W. J., Muller, C. D., and Eugui, E. M. (1993) Ann. N. Y. Acad. Sci. 696, 63-87 [Medline] [Order article via Infotrieve]
  4. DeClerq, E. (1993) Adv. Virus Res. 42, 1-55 [Medline] [Order article via Infotrieve]
  5. Hupe, D. J., Azzolina, B. A., and Behrens, N. D. (1986) J. Biol. Chem. 261, 8363-8369 [Abstract/Free Full Text]
  6. Verham, R., Meek, T. D., Hedstrom, L., and Wang, C. C. (1987) Mol. Biochem. Parasitol. 24, 1-12 [CrossRef][Medline] [Order article via Infotrieve]
  7. Wang, W., Papov, V. V., Minakawa, N., Matsuda, A., Biemann, K., and Hedstrom, L. (1996) Biochemistry 35, 95-101 [CrossRef][Medline] [Order article via Infotrieve]
  8. Collart, F. R., and Huberman, E. (1988) J. Biol. Chem. 263, 15769-15772 [Abstract/Free Full Text]
  9. Natsumeda, Y., Ohno, S., Kawasaki, H., Konno, Y., Weber, G., and Suzuki, K. (1990) J. Biol. Chem. 265, 5292-5295 [Abstract/Free Full Text]
  10. Collart, F. R., Chubb, C. B., Mirkin, B. L., and Huberman, E. (1992) Cancer Res. 52, 5826-5828 [Abstract]
  11. Nagai, M., Natsumeda, Y., and Weber, G. (1992) Cancer Res. 52, 258-261 [Abstract]
  12. Link, J. O., and Straub, K. (1996) J. Am. Chem. Soc. 118, 2091-2092 [CrossRef]
  13. Huete-Perez, J. A., Wu, J. C., Witby, F. G., and Wang, C. C. (1995) Biochemistry 34, 13889-13894 [Medline] [Order article via Infotrieve]
  14. Franklin, T., and Cook, J. (1969) Biochem. J. 113, 515-524 [Medline] [Order article via Infotrieve]
  15. Wu, J. C. (1994) Perspect. Drug Discov. Des. 2, 185-204
  16. Wu, T., and Scrimgeour, K. G. (1973) Can. J. Biochem. 51, 1391-1398 [Medline] [Order article via Infotrieve]
  17. Hedstrom, L., and Wang, C. C. (1990) Biochemistry 29, 849-554 [Medline] [Order article via Infotrieve]
  18. Carr, S. F., Papp, E., Wu, J. C., and Natsumeda, Y. (1993) J. Biol. Chem. 268, 27286-27290 [Abstract/Free Full Text]
  19. Fleming, M. A., Chambers, S. P., Connelly, P. R., Nimmesgern, E., Fox, T., Bruzzese, F. J., Hoe, S. T., Fulghum, J. R., Livingston, D. J., Stuver, C. M., Sintchak, M. D., Wilson, K. P., and Thomson, J. A. (1996) Biochemistry 35, 6990-6997 [CrossRef][Medline] [Order article via Infotrieve]
  20. Sintchak, M. D., Fleming, M. A., Futer, O., Raybuck, S. A., Chambers, S. P., Caron, P. R., Murcko, M., and Wilson, K. P. (1996) Cell 85, 921-930 [Medline] [Order article via Infotrieve]
  21. Teideman, A. A., and Smith, J. M. (1985) Nucleic Acids Res. 13, 1303-1316 [Abstract]
  22. Kanzaki, N., and Miyagawa, K. (1990) Nucleic Acids Res. 18, 6710 [Medline] [Order article via Infotrieve]
  23. Beck, J. T., Zhao, S., and Wang, C. C. (1994) Exp. Parasitol. 78, 101-112 [CrossRef][Medline] [Order article via Infotrieve]
  24. Collart, F. R., and Huberman, E. (1987) Mol. Cell. Biol. 7, 3328-3331 [Medline] [Order article via Infotrieve]
  25. Wilson, K., Collart, F., Huberman, E., Stringer, J., and Ullman, B. (1991) J. Biol. Chem. 266, 1665-1671 [Abstract/Free Full Text]
  26. Wilson, K., Berens, R. L., Sifri, C. D., and Ullman, B. (1994) J. Biol. Chem. 269, 28979-28987 [Abstract/Free Full Text]
  27. Ullman, B. (1983) J. Biol. Chem. 258, 523-528 [Abstract/Free Full Text]
  28. Cohen, M. B. (1987) Som. Cell Mol. Genet. 13, 627-633 [Medline] [Order article via Infotrieve]
  29. Lightfoot, T., and Snyder, F. F. (1994) Biochim. Biophys. Acta 1217, 156-162 [Medline] [Order article via Infotrieve]
  30. Hedstrom, L., Cheung, K., and Wang, C. C. (1990) Biochem. Pharmacol. 39, 151-160 [Medline] [Order article via Infotrieve]
  31. Scheidel, L. M., and Stollar, V. (1991) Virology 181, 490-499 [Medline] [Order article via Infotrieve]
  32. Franklin, T. J., Jacobs, V., Jones, G., Ple, P., and Bruneau, P. (1996) Cancer Res. 56, 984-987 [Abstract]
  33. Hedstrom, L., Szilagyi, L., and Rutter, W. J. (1992) Science 255, 1249-1253 [Medline] [Order article via Infotrieve]
  34. Kunkle, T. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 488-492 [Abstract]
  35. Glesne, D. A., and Huberman, E. (1994) Biochem. Biophys. Res. Commun. 205, 537-544 [CrossRef][Medline] [Order article via Infotrieve]
  36. Zimmermann, A. G., Spychala, J., and Mitchell, B. S. (1995) J. Biol. Chem. 270, 6808-6814 [Abstract/Free Full Text]
  37. Amann, E., and Brosius, J. (1985) Gene (Amst.) 40, 183-190 [CrossRef][Medline] [Order article via Infotrieve]
  38. Antonino, L. C., and Wu, J. C. (1994) Biochemistry 33, 1753-1759 [Medline] [Order article via Infotrieve]
  39. Hager, P. W., Collart, F. R., Huberman, E., and Mitchell, B. S. (1995) Biochem. Pharmacol. 49, 1323-1329 [CrossRef][Medline] [Order article via Infotrieve]
  40. Schaaper, R. M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8126-8130 [Abstract]
  41. Nijkamp, H. J. J., and De Haan, P. G. (1967) Biochim. Biophys. Acta 145, 31-40 [Medline] [Order article via Infotrieve]
  42. Ikegami, T., Natsumeda, Y., and Weber, G. (1987) Life Sci. 40, 2277-2282 [CrossRef][Medline] [Order article via Infotrieve]
  43. Greco, W. R., and Hakala, M. T. (1979) J. Biol. Chem. 254, 12104-12109 [Medline] [Order article via Infotrieve]
  44. Morrison, J. F. (1969) Biochim. Biophys. Acta 185, 269-286 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.