Monovalent Cation Activation in Escherichia coli Inosine 5′-Monophosphate Dehydrogenase

Kerr, Kathleen; Cahoon, M.; Bosco, Daryl A.; Hedstrom, Lizbeth

doi:10.1006/abbi.1999.1644

Cited by 29 publications

(31 citation statements)

References 22 publications

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“…A large peak of negative difference density was observed when K ϩ was modeled as well as high B factors. Because of the high concentration of sodium in the crystallization buffer, the previous observation that a sodium ion binds competitively with K ϩ in microbial IMPDH (33), and B factors that are near those of the neighboring atoms, a Na ϩ ion was placed in the final model (Fig. 7).…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of Tritrichomonas foetus Inosine Monophosphate Dehydrogenase in Complex with the Inhibitor Ribavirin Monophosphate Reveals a Catalysis-dependent Ion-binding Site

Prosise

Wu²,

Luecke

2002

Journal of Biological Chemistry

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Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in GMP biosynthesis. The resulting intracellular pool of guanine nucleotides is of great importance to all cells for use in DNA and RNA synthesis, metabolism, and signal transduction. The enzyme binds IMP and the cofactor NAD ؉ in random order, IMP is converted to XMP, NAD ؉ is reduced to NADH, and finally, NADH and then XMP are released sequentially. XMP is subsequently converted into GMP by GMP synthetase. Drugs that decrease GMP synthesis by inhibiting IMPDH have been shown to have antiproliferative as well as antiviral activity. Several drugs are in use that target the substrate-or cofactor-binding site; however, due to differences between the mammalian and microbial isoforms, most drugs are far less effective against the microbial form of the enzyme than the mammalian form. The high resolution crystal structures of the protozoan parasite Tritrichomonas foetus IMPDH complexed with the inhibitor ribavirin monophosphate as well as monophosphate together with a second inhibitor, mycophenolic acid, are presented here. These structures reveal an active site cation identified previously only in the Chinese hamster IMPDH structure with covalently bound IMP. This cation was not found previously in apo IMPDH, IMPDH in complex with XMP, or covalently bound inhibitor,indicatingthatthecation-bindingsitemaybecatalysisdependent. A comparison of T. foetus IMPDH with the Chinese hamster and Streptococcus pyogenes structures reveals differences in the active site loop architecture, which contributes to differences in cation binding during the catalytic sequence and the kinetic rates between bacterial, protozoan, and mammalian enzymes. Exploitation of these differences may lead to novel inhibitors, which favor the microbial form of the enzyme. Inosine-5Ј-monophosphate dehydrogenase (IMPDH)1 (E.C. 1.1.1.205) is the enzyme that catalyzes the NAD ϩ -dependent oxidation reaction that converts inosine monophosphate (IMP) to xanthosine monophosphate (XMP). This is the rate-limiting step in guanine nucleotide biosynthesis. The kinetic mechanism for both mammalian and microbial IMPDH has been extensively studied (1-3), and the crystal structures have been determined for two bacterial, two mammalian, and one protozoan form of IMPDH (4 -9). The enzymatic reaction appears to follow a random-in ordered-out kinetic mechanism where the substrate IMP and cofactor NAD ϩ bind to the enzyme active site followed by the nucleophilic attack on the C2 position of IMP by the active site cysteine to form E-IMP covalent intermediate. Hydride transfer from the E-IMP intermediate to NADϩ results in the formation of E-XMP intermediate and reduced NADH. A water molecule in the active site helps to hydrolyze the covalent intermediate and to release XMP only after the cofactor NADH has dissociated from the active site. GMP, the next product in purine biosynthesis, supplies an intracellular pool of guanine nucleotide for DNA and RNA synthesis, is used in energy storage, and plays a...

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Section: Resultsmentioning

confidence: 99%

Crystal Structure of Tritrichomonas foetus Inosine Monophosphate Dehydrogenase in Complex with the Inhibitor Ribavirin Monophosphate Reveals a Catalysis-dependent Ion-binding Site

Prosise

Wu²,

Luecke

2002

Journal of Biological Chemistry

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“…IMPDH, encoded by the guaB gene catalyses the reversible conversion of IMP to XMP. In E. coli, at least two different enzymic pathways lead to the synthesis of XMP (Neuhard & Nygaard, 1996;Pimkin et al, 2009) which are well characterized (Gilbert et al, 1979;Gilbert & Drabble, 1980;Kerr & Hedstrom, 1997;Kerr et al, 2000;Tiedeman & Smith, 1985). However, the M. tuberculosis H37Rv purine de novo biosynthesis pathway enzymes have received little attention to date and are poorly studied.…”

Section: Discussionmentioning

confidence: 99%

“…Monovalent cations, such as K + , activate IMPDH (Hedstrom, 2009;Kerr et al, 2000;Zhou et al, 1997) and in a similar fashion Mt-GuaB2 exhibited a requirement for KCl (Fig. 3a).…”

Section: Biochemical Properties Of Recombinant Mt-guab Orthologuesmentioning

confidence: 99%

Identification of novel diphenyl urea inhibitors of Mt-GuaB2 active against Mycobacterium tuberculosis

et al. 2011

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In contrast with most bacteria, which harbour a single inosine monophosphate dehydrogenase (IMPDH) gene, the genomic sequence of Mycobacterium tuberculosis H37Rv predicts three genes encoding IMPDH: guaB1, guaB2 and guaB3. These three genes were cloned and expressed in Escherichia coli to evaluate functional IMPDH activity. Purified recombinant Mt-GuaB2, which uses inosine monophosphate as a substrate, was identified as the only active GuaB orthologue in M. tuberculosis and showed optimal activity at pH 8.5 and 37 6C. Mt-GuaB2 was inhibited significantly in vitro by a panel of diphenyl urea-based derivatives, which were also potent anti-mycobacterial agents against M. tuberculosis and Mycobacterium smegmatis, with MICs in the range of 0.2-0.5 mg ml "1 . When Mt-GuaB2 was overexpressed on a plasmid in trans in M. smegmatis, a diphenyl urea analogue showed a 16-fold increase in MIC. Interestingly, when Mt-GuaB orthologues (Mt-GuaB1 and 3) were also overexpressed on a plasmid in trans in M. smegmatis, they also conferred resistance, suggesting that although these Mt-GuaB orthologues were inactive in vitro, they presumably titrate the effect of the inhibitory properties of the active compounds in vivo.

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“…In the absence of K + , multiple conformers are present that bind with different affinities for Pdx r , giving rise to complex behavior and splitting of resonances in the HSQC experiment. Such allosteric regulation has been observed in other enzymes, including IMPDH (25,26), pyruvate kinase (27)(28)(29) and dialkylglycine decarboxylase (30,31).…”

Section: Antagonistic Behavior In K + and Effector Binding By Cyp-s-comentioning

confidence: 98%

Specific Effects of Potassium Ion Binding on Wild-Type and L358P Cytochrome P450cam

et al. 2006

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The camphor monoxygenase cytochrome P450 cam (CYP101) requires potassium ion (K + ) to drive formation of the characteristic high-spin state of the heme Fe +3 upon substrate binding. Amide 1 H, 15 N correlations in perdeuterated [U-15 N] CYP101 were monitored as a function of K + concentration by 2D-TROSY-HSQC in both camphor-bound oxidized (CYP-S) and camphor-and CO-bound reduced CYP101 (CYP-S-CO). In both forms, K + -induced spectral perturbations are detected in the vicinity of the K + binding site proposed from crystallographic structures, but are larger and more widespread structurally in CYP-S than in CYP-S-CO. In CYP-S-CO, K + -induced perturbations occur primarily near the proposed K + binding site in the B-B' loop and B' helix, which are also perturbed by binding of effector, putidaredoxin (Pdx). The spectral effects of K + binding in CYP-S-CO oppose those observed upon Pdx r titration. However, Pdx r titration of CYP-S-CO in the absence of K + results in multiple conformations. The spin-state equilibrium in the L358P mutant of CYP101 is more sensitive to K + concentration than WT CYP101, consistent with a hypothesis that L358P preferentially populates conformations enforced by Pdx binding in WT CYP101. Thallium (I), a K + mimic, minimizes the effects of Pdx titration on the NMR spectrum of CYP-S-CO, but is competent to replace K + in driving the formation of high-spin CYP-S. These observations suggest that the role of K + is to stabilize conformers of CYP-S that drive the spin state change prior to the first electron transfer, and that K + stabilizes the CYP-S-CO conformer that interacts with Pdx. However, upon binding of Pdx, further conformational changes occur that disfavor K + binding.

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Monovalent Cation Activation in Escherichia coli Inosine 5′-Monophosphate Dehydrogenase

Cited by 29 publications

References 22 publications

Crystal Structure of Tritrichomonas foetus Inosine Monophosphate Dehydrogenase in Complex with the Inhibitor Ribavirin Monophosphate Reveals a Catalysis-dependent Ion-binding Site

Crystal Structure of Tritrichomonas foetus Inosine Monophosphate Dehydrogenase in Complex with the Inhibitor Ribavirin Monophosphate Reveals a Catalysis-dependent Ion-binding Site

Identification of novel diphenyl urea inhibitors of Mt-GuaB2 active against Mycobacterium tuberculosis

Specific Effects of Potassium Ion Binding on Wild-Type and L358P Cytochrome P450cam

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