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. Inosine 5Ј-monophosphate dehydrogenase catalyzes the conversion of IMP to XMP 1 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 IMPDHdeficient 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 cry...
The conversion of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) is the committed and rate-limiting reaction in de novo guanine nucleotide biosynthesis. Inosine 5'- monophosphate dehydrogenase (IMPDH) is the enzyme that catalyzes the oxidation of IMP to XMP with the concomitant reduction of nicotinamide adenine dinucleotide (from NAD(+) to NADH). Because of its critical role in purine biosynthesis, IMPDH is a drug design target for anticancer, antiinfective, and immunosuppressive chemotherapy. We have determined the crystal structure of IMPDH from Borrelia burgdorferi, the bacterial spirochete that causes Lyme disease, with a sulfate ion bound in the IMP phosphate binding site. This is the first structure of IMPDH in the absence of substrate or cofactor where the active-site loop (loop 6), which contains the essential catalytic residue Cys 229, is clearly defined in the electron density. We report that a seven residue region of loop 6, including Cys229, is tilted more than 6 A away from its position in substrate- or substrate analogue-bound structures of IMPDH, suggestive of a conformational change. The location of this loop between beta6 and alpha6 links IMPDH to a family of beta/alpha barrel enzymes known to utilize this loop as a functional lid during catalysis. Least-squares minimization, root-mean-square deviation analysis, and inspection of the molecular surface of the loop 6 region in the substrate-free B. burgdorferi IMPDH and XMP-bound Chinese hamster IMPDH show that loop 6 follows a similar pattern of hinged rigid-body motion and indicates that IMPDH may be using loop 6 to bind and sequester substrate and to recruit an essential catalytic residue.
-Nous utilisons des champs magn étiques pour orienter des cristaux liquides nématiques ainsi que des solutions cristallines colloïdales monodisperses de virus du tabac (TMV). Les instabilités hydrodynamiques induites par les réorientations sous champ magnétique ont été étudiées sur des cristaux liquides nématiques bien orientés. Abstract-Magnetic fields have been used to produce well aligned nematic liquid crystalline and colloidal crystalline samples of nearly monodisperse tobacco mosaic virus (TMV). Hydrodynamic instabilities induced by reorientation in a magnetic field have been studied in the well aligned nematic liquid crystalline samples.
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 Gln 277 3 Arg causes a 9-fold increase in the K i of MPA, a 5-6-fold increase in the K m values for IMP and NAD, and a 3-fold decrease in k cat relative to wild type. The mutation of Ala 462 3 Thr causes a 3-fold increase in the K i for MPA, a 2.5-fold increase in the K m for NAD, and a 1.5-fold increase in k cat . The combination of these two mutations does not increase the K i for MPA, but does increase the K m for NAD 3-fold relative to Q277R and restores k cat to wild type levels. Q277R/A462T is the first human IMPDH mutant with increased K i for MPA and wild type activity. The third mutant IMPDH contains two mutations, Phe 465 3 Ser and Asp 470 3 Gly. K i for MPA is increased 3-fold in this mutant enzyme, and K m for IMP is also increased 3-fold, while the K m for NAD and k cat are unchanged. Thus increases in the K i for MPA do not correlate with changes in K m for either IMP or NAD, nor to changes in k cat . 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 (2-4). 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 (5-7). Two human IMPDH isozymes exist; type I is constitutively expressed, while type II is expressed in rapidly proliferating cells (8 -11). The IMPDH reaction involves attack of Cys 331 (human type II numbering) at the 2-position of IMP, followed by expulsion of the hydride to NAD (Fig. 1) (Fig. 1), and the crystal structure of the E-XMP⅐MPA complex of IMPDH from Chinese hamster has recently been solved (12,19,20 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 -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 no...
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