Expression, Purification, and Characterization of Inosine 5′-Monophosphate Dehydrogenase from Borrelia burgdorferi

Zhou, Xun; Cahoon, M.; Rosa, Patricia A.; Hedstrom, Lizbeth

doi:10.1074/jbc.272.35.21977

Cited by 50 publications

(67 citation statements)

References 32 publications

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“…1). Moreover, it has been demonstrated that the GuaB and GuaA enzymes, the final two enzymes in the B. burgdorferi purine salvage pathway required for guanine nucleotide synthesis (28,67), are able to use dIMP and dXMP, respectively, as substrates to produce dGMP (23), in addition to the well-characterized substrates IMP and XMP, respectively, to generate GMP (28,67) (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

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GuaA and GuaB Are Essential forBorrelia burgdorferiSurvival in the Tick-Mouse Infection Cycle

et al. 2009

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Pathogens lacking the enzymatic pathways for de novo purine biosynthesis are required to salvage purines and pyrimidines from the host environment for synthesis of DNA and RNA. Two key enzymes in purine salvage pathways are IMP dehydrogenase (GuaB) and GMP synthase (GuaA), encoded by the guaB and guaA genes, respectively. While these genes are typically found on the chromosome in most bacterial pathogens, the guaAB operon of Borrelia burgdorferi is present on plasmid cp26, which also harbors a number of genes critical for B. burgdorferi viability. Using molecular genetics and an experimental model of the tick-mouse infection cycle, we demonstrate that the enzymatic activities encoded by the guaAB operon are essential for B. burgdorferi mouse infectivity and provide a growth advantage to spirochetes in the tick. These data indicate that the GuaA and GuaB proteins are critical for the survival of B. burgdorferi in the infection cycle and highlight a potential difference in the requirements for purine salvage in the disparate mammalian and tick environments.Purine metabolism is critical for the growth and virulence in mammals of many bacterial pathogens (11,26,29,33,51). Borrelia burgdorferi, the infectious agent of Lyme borreliosis, lacks the genes encoding the enzymes required for de novo nucleotide synthesis (8, 12) and therefore must rely on salvage of purines and pyrimidines from its hosts for nucleic acid biosynthesis (21, 35). Furthermore, B. burgdorferi lacks the genes encoding key enzymes required for a classic purine salvage pathway, including hpt (hypoxanthine-guanine phosphoribosyltransferase), purA (adenylosuccinate synthase), purB (adenylosuccinate lyase), and the locus encoding a ribonucleotide reductase (4,8,12,35,66). Despite the absence of a ribonucleotide reductase, an enzyme critical for the generation of deoxynucleotides through enzymatic reduction of ribonucleotides (32), a novel purine salvage pathway that involves salvage of deoxynucleosides from the host and interconversion of purine bases to deoxynucleosides by BB0426, a deoxyribosyl transferase, has recently been demonstrated for B. burgdorferi (23) (Fig. 1).In its infection cycle, B. burgdorferi passages between two disparate environments with potentially distinct purine availabilities, the tick vector and a mammalian host. Hypoxanthine is the most abundant purine in mammalian blood (17), and it is available for salvage by B. burgdorferi during the blood meal of an infected tick and during the spirochete's transient presence in the mammalian bloodstream. Despite the absence of the hpt gene, we and others have shown that B. burgdorferi is able to transport and incorporate low levels of hypoxanthine (23,35). During mammalian infection B. burgdorferi resides in various tissues, including the skin, heart, bladder, and joints. Adenine has been shown to be ubiquitous in mammalian tissues (61) and therefore is available for salvage by B. burgdorferi. Guanine is present at low levels in mammalian blood and tissues (17, 61); however, the amount m...

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Section: Resultsmentioning

confidence: 99%

“…The enzymes required for the final two steps of guanine nucleotide biosynthesis, IMPDH and GMP synthase, are encoded by the guaB and guaA genes, respectively. The guaA and guaB genes and the corresponding activities of their protein products are conserved in B. burgdorferi (28,67). These genes are typically carried on the chromosomes of bacterial species.…”

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GuaA and GuaB Are Essential forBorrelia burgdorferiSurvival in the Tick-Mouse Infection Cycle

et al. 2009

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“…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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“…Proposed or known functions have also been determined for chbC (50), oppAIV (7), and guaB (30,51,53) (Fig. 1), all of which have been successfully inactivated without inhibition of in vitro growth.…”

Section: Discussionmentioning

confidence: 99%

The Essential Nature of the Ubiquitous 26-Kilobase Circular Replicon ofBorrelia burgdorferi

2004

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The genome of the type strain (B31) of Borrelia burgdorferi, the causative agent of Lyme disease, is composed of 12 linear and 9 circular plasmids and a linear chromosome. Plasmid content can vary among strains, but one 26-kb circular plasmid (cp26) is always present. The ubiquitous nature of cp26 suggests that it provides functions required for bacterial viability. We tested this hypothesis by attempting to selectively displace cp26 with an incompatible but replication-proficient vector, pBSV26. While pBSV26 transformants contained this incompatible vector, the vector coexisted with cp26, which is consistent with the hypothesis that cp26 carries essential genes. Several cp26 genes with ascribed or predicted functions may be essential. These include the BBB29 gene, which has sequence homology to a gene encoding a glucose-specific phosphotransferase system component, and the resT gene, which encodes a telomere resolvase involved in resolution of the replicated telomeres of the linear chromosome and plasmids. The BBB29 gene was successfully inactivated by allelic exchange, but attempted inactivation of resT resulted in merodiploid transformants, suggesting that resT is required for B. burgdorferi growth. To determine if resT is the only cp26 gene essential for growth, we introduced resT into B. burgdorferi on pBSV26. This did not result in displacement of cp26, suggesting that additional cp26 genes encode vital functions. We concluded that B. burgdorferi plasmid cp26 encodes functions critical for survival and thus shares some features with the chromosome.

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Expression, Purification, and Characterization of Inosine 5′-Monophosphate Dehydrogenase from Borrelia burgdorferi

Cited by 50 publications

References 32 publications

GuaA and GuaB Are Essential forBorrelia burgdorferiSurvival in the Tick-Mouse Infection Cycle

GuaA and GuaB Are Essential forBorrelia burgdorferiSurvival in the Tick-Mouse Infection Cycle

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

The Essential Nature of the Ubiquitous 26-Kilobase Circular Replicon ofBorrelia burgdorferi

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