Crystal Structure of<i>Tritrichomonas foetus</i>Inosine-5‘-monophosphate Dehydrogenase and the Enzyme−Product Complex

The protozoan parasite Cryptosporidium parvum causes severe enteritis with substantial morbidity and mortality among AIDS patients and young children. No fully effective treatment is available. C. parvum relies on inosine 5-monophosphate dehydrogenase (IMPDH) to produce guanine nucleotides and is highly susceptible to IMPDH inhibition. Furthermore, C. parvum obtained its IMPDH gene by lateral transfer from an epsilon-proteobacterium, suggesting that the parasite enzyme might have very different characteristics than the human counterpart. Here we describe the expression of recombinant C. parvum IMPDH in an Escherichia coli strain lacking the bacterial homolog. Expression of the parasite gene restores growth of this mutant on minimal medium, confirming that the protein has IMPDH activity. The recombinant protein was purified to homogeneity and used to probe the enzyme's mechanism, structure, and inhibition profile in a series of kinetic experiments. The mechanism of the C. parvum enzyme involves the random addition of substrates and ordered release of products with rate-limiting hydrolysis of a covalent enzyme intermediate. The pronounced resistance of C. parvum IMPDH to mycophenolic acid inhibition is in strong agreement with its bacterial origin. The values of K m for NAD and K i for mycophenolic acid as well as the synergistic interaction between tiazofurin and ADP differ significantly from those of the human enzymes. These data suggest that the structure and dynamic properties of the NAD binding site of C. parvum IMPDH can be exploited to develop parasitespecific inhibitors.

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“…5). This hypothesis is supported by the varying extents of disorder in this loop in structures of IMPDHs from different sources (39,43,44).…”

Section: Discussionmentioning

confidence: 84%

Cryptosporidium parvum IMP Dehydrogenase

Umejiego¹,

Li²,

Riera

et al. 2004

Journal of Biological Chemistry

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“…We also show that genetic complementation is feasible across species boundaries among Apicomplexa. Using this heterologous complementation, we have cloned a Cryptosporidium IMPDH gene involved in purine salvage; this pathway is essential to parasite survival and therefore is actively pursued for drug development (38)(39)(40)(41). The finding that the parasite has obtained this gene by lateral gene transfer from a bacterial source might increase its potential as a target for drug development (41,42 …”

Section: Resultsmentioning

confidence: 99%

Genetic complementation in apicomplexan parasites

Striepen

White

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

120

110

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A robust forward genetic model for Apicomplexa could greatly enhance functional analysis of genes in these important protozoan pathogens. We have developed and successfully tested a genetic complementation strategy based on genomic insertion in Toxoplasma gondii. Adapting recombination cloning to genomic DNA, we show that complementing sequences can be shuttled between parasite genome and bacterial plasmid, providing an efficient tool for the recovery and functional assessment of candidate genes. We show complementation, gene cloning, and biological verification with a mutant parasite lacking hypoxanthine-xanthine-guanine phosphoribosyltransferase and a T. gondii cDNA library. We also explored the utility of this approach to clone genes based on function from other apicomplexan parasites using Toxoplasma as a surrogate. A heterologous library containing Cryptosporidium parvum genomic DNA was generated, and we identified a C. parvum gene coding for inosine 5-monophosphate-dehydrogenase (IMPDH). Interestingly, phylogenetic analysis demonstrates a clear eubacterial origin of this gene and strongly suggests its lateral transfer from -proteobacteria. The prokaryotic origin of this enzyme might make it a promising target for therapeutics directed against Cryptosporidium.purine salvage ͉ lateral gene transfer ͉ Cryptosporidium parvum ͉ Toxoplasma gondii T he phylum Apicomplexa includes a large number of obligate intracellular parasites, among which are the human pathogens Plasmodium (malaria), Toxoplasma (AIDS-related encephalitis), Cryptosporidium, and Cyclospora (severe enteritis) as well as many parasites of substantial veterinary importance (Eimeria, Theileria, Sarcocystis, and Babesia). Toxoplasma gondii has emerged as a versatile genetic model organism to study the basic biology of this group of parasites (1). T. gondii is safe and easy to culture in vitro, and excellent animal models are available; it is also amendable to molecular genetic experimentation. The high transfection efficiencies, which can be achieved in this organism, have spurred the development of a powerful set of reverse genetic tools (2-4).Apicomplexa, as with most protists, diverged relatively early in the eukaryotic lineage and have many biological features not shared by major eukaryotic model systems (e.g., intracellular parasitism or the possession of secondary plastids). Several large scale sequencing efforts have increased the number of known apicomplexan genes drastically (5). Assigning biological functions to many of these genes, however, remains a major challenge. A forward genetic approach could bridge this gap and permit functional analysis of genes unique to Apicomplexa. The parasites maintain a haploid genome over most of their life cycle, which should greatly facilitate the generation of loss of function mutants. Indeed chemical as well as insertional mutagenesis has been applied successfully in several screens targeting nonessential genes involved in nucleotide biosynthesis (6-11). Parasites harboring conditional temperature-sens...

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“…deviation of 2.3 Å for 213 equivalent C␣ positions, a Z-score of 21.0, and a sequence identity of 21%) (Fig. S2A) (30). The second highest Z-score is obtained with human guanosine monophosphate reductase (unpublished deposition; PDB code 2BLE; an r.m.s.…”

Section: Respectively) D Figure Of Merit ϭ ͉͗⌺P(␣)ementioning

confidence: 94%

Crystal Structure of 2-Nitropropane Dioxygenase Complexed with FMN and Substrate

Ha¹,

Min

Lee

et al. 2006

Journal of Biological Chemistry

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Nitroalkane compounds are widely used in chemical industry and are also produced by microorganisms and plants. Some nitroalkanes have been demonstrated to be carcinogenic, and enzymatic oxidation of nitroalkanes is of considerable interest. 2-Nitropropane dioxygenases from Neurospora crassa and Williopsis mrakii (Hansenula mrakii), members of one family of the nitroalkane-oxidizing enzymes, contain FMN and FAD, respectively. The enzymatic oxidation of nitroalkanes by 2-nitropropane dioxygenase operates by an oxidase-style catalytic mechanism, which was recently shown to involve the formation of an anionic flavin semiquinone. This represents a unique case in which an anionic flavin semiquinone has been experimentally observed in the catalytic pathway for oxidation catalyzed by a flavin-dependent enzyme. Here we report the first crystal structure of 2-nitropropane dioxygenase from Pseudomonas aeruginosa in two forms: a binary complex with FMN and a ternary complex with both FMN and 2-nitropropane. The structure identifies His 152 as the proposed catalytic base, thus providing a structural framework for a better understanding of the catalytic mechanism.Nitroalkanes are widely used in industry, because they are useful as intermediate compounds in chemical synthesis (1, 2). They are also synthesized by various organisms. Many antibiotics, e.g. chloramphenicol and azomycin, contain nitro groups, and many leguminous plants produce nitro toxins such as 3-nitro-1-propionic acid and 3-nitro-1-propanol (3). However, many nitroalkanes are expected to be toxic, and some have been shown to be carcinogenic (4 -10). For example, 2-nitropropane causes the formation of both 8-hydroxy-and 8-aminoguanine in the DNA and RNA (11). The enzymatic oxidation of nitroalkanes into less toxic species can therefore be exploited for use in bioremediation.2-Nitropropane dioxygenase (EC 1.13.11.32), one of the nitroalkaneoxidizing enzyme families, catalyzes oxidative denitrification of nitroalkanes to their corresponding carbonyl compounds and nitrites. To date, 2-nitropropane dioxygenase has been isolated from a fungus Neurospora crassa (12) and a yeast Williopsis mrakii (Hansenula mrakii) (13).The two enzymes have similar molecular masses of ϳ40 kDa, but their prosthetic groups are different. FMN and FAD are found in the N. crassa and W. mrakii (H. mrakii) enzymes, respectively (14, 15). The ncd-2 gene encoding for 2-nitropropane dioxygenase in N. crassa has been cloned and expressed in Escherichia coli (16). The heterologously expressed enzyme was found to be a homodimer containing 1 mol of non-covalently bound FMN per mole of subunit (16). A steady-state kinetic analysis showed that the preferred substrates for the enzyme are anionic nitronates as compared with neutral nitroalkanes and that the enzyme has broad substrate specificity that is independent of substrate size (16).It has been shown that 2-nitropropane dioxygenase operates through an oxidase-style catalytic mechanism, in which substrate oxidation occurs prior to and independently ...

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Crystal Structure ofTritrichomonas foetusInosine-5‘-monophosphate Dehydrogenase and the Enzyme−Product Complex

Cited by 53 publications

References 34 publications

Cryptosporidium parvum IMP Dehydrogenase

Cryptosporidium parvum IMP Dehydrogenase

Genetic complementation in apicomplexan parasites

Crystal Structure of 2-Nitropropane Dioxygenase Complexed with FMN and Substrate

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