Mapping of a new <i>pan</i> mutation in <i>Escherichia coli</i> K-12

Manch, Jean N.

doi:10.1139/m81-190

Cited by 6 publications

(6 citation statements)

References 7 publications

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“…These mutations defined the panE locus and caused pantothenate auxotrophy only in strains defective for acetohydroxy acid isomeroreductase (IlvC) (10). Similar mutations were isolated in Salmonella typhimurium, and crude extracts of the panE ilvC double mutant had no ketopantoic acid reductase activity, consistent with the nutri-tional requirement of the strain (13,16). From this work, it was suggested that both IlvC and PanE have ketopantoic acid reductase activity and that together they account for 100% of the cellular activity.…”

supporting

confidence: 67%

The panE Gene, Encoding Ketopantoate Reductase, Maps at 10 Minutes and Is Allelic to apbA in Salmonella typhimurium

Frodyma

Downs

1998

J Bacteriol

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In Salmonella typhimurium, precursors to the pyrimidine moiety of thiamine are synthesized de novo by the purine biosynthetic pathway or the alternative pyrimidine biosynthetic (APB) pathway. TheapbA gene was the first locus defined as required for function of the APB pathway (D. M. Downs and L. Petersen, J. Bacteriol. 176:4858–4864, 1994). Recent work showed the ApbA protein catalyzes the NADPH-specific reduction of ketopantoic acid to pantoic acid. This activity had previously been associated with the pantothenate biosynthetic gene panE. Although previous reports placed panE at 87 min on the Escherichia coli chromosome, we show herein that apbA andpanE are allelic and map to 10 min on both the S. typhimurium and E. coli chromosomes. Results presented here suggest that the role of ApbA in thiamine synthesis is indirect since in vivo labeling studies showed that pantoic acid, the product of the ApbA-catalyzed reaction, is not a direct precursor to thiamine via the APB pathway.

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supporting

confidence: 67%

The panE Gene, Encoding Ketopantoate Reductase, Maps at 10 Minutes and Is Allelic to apbA in Salmonella typhimurium

Frodyma

Downs

1998

J Bacteriol

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“…The pantothenate biosynthetic pathway is present in some plants, fungi, bacteria, and archaea, making the pathway a strong candidate for the discovery of novel antibiotics (1). The pantothenate biosynthesis pathway was first genetically and chemically defined in Escherichia coli and Salmonella enterica serovar Typhimurium and is composed of four enzymes that use the precursors 2-oxoisovalerate and L-aspartic acid to make pantothenate (2,3) (Fig. 1A).…”

mentioning

confidence: 99%

PanG, a New Ketopantoate Reductase Involved in Pantothenate Synthesis

Miller

LoVullo

Kijek

et al. 2012

Journal of Bacteriology

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Pantothenate, commonly referred to as vitamin B 5 , is an essential molecule in the metabolism of living organisms and forms the core of coenzyme A. Unlike humans, some bacteria and plants are capable of de novo biosynthesis of pantothenate, making this pathway a potential target for drug development. Francisella tularensis subsp. tularensis Schu S4 is a zoonotic bacterial pathogen that is able to synthesize pantothenate but is lacking the known ketopantoate reductase (KPR) genes, panE and ilvC, found in the canonical Escherichia coli pathway. Described herein is a gene encoding a novel KPR, for which we propose the name panG (FTT1388), which is conserved in all sequenced Francisella species and is the sole KPR in Schu S4. Homologs of this KPR are present in other pathogenic bacteria such as Enterococcus faecalis, Coxiella burnetii, and Clostridium difficile. Both the homologous gene from E. faecalis V583 (EF1861) and E. coli panE functionally complemented Francisella novicida lacking any KPR. Furthermore, panG from F. novicida can complement an E. coli KPR double mutant. A Schu S4 ⌬panG strain is a pantothenate auxotroph and was genetically and chemically complemented with panG in trans or with the addition of pantolactone. There was no virulence defect in the Schu S4 ⌬panG strain compared to the wild type in a mouse model of pneumonic tularemia. In summary, we characterized the pantothenate pathway in Francisella novicida and F. tularensis and identified an unknown and previously uncharacterized KPR that can convert 2-dehydropantoate to pantoate, PanG.

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“…The panE gene encoding KPR remained elusive, however, due in part to the fact that an enzyme of valine and isoleucine biosynthesis, acetohydroxyacid isomeroreductase (AHIR, EC 1.1.1.86), encoded by ilVC, can catalyze the same reaction as KPR, albeit less efficiently. Mutants of ilVC still retain KPR activity, whereas ilVC panE double mutants have no measurable enzyme activity and are pantoate auxotrophs (3,4). The panE gene from S. typhimurium has recently been shown to be allelic to the apbA gene (5).…”

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confidence: 99%

Crystal Structure of Escherichia coli Ketopantoate Reductase at 1.7 Å Resolution and Insight into the Enzyme Mechanism

et al. 2001

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Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes the NADPH-dependent reduction of ketopantoate to pantoate on the pantothenate (vitamin B5) biosynthetic pathway. The Escherichia coli panE gene encoding KPR was cloned and expressed at high levels as the native and selenomethionine-substituted (SeMet) proteins. Both native and SeMet recombinant proteins were purified by three chromatographic steps, to yield pure proteins. The wild-type enzyme was found to have a K M(NADPH) of 20 μM, a K M(ketopantoate) of 60 μM, and a k cat of 40 s-1. Regular prismatic KPR crystals were prepared using the hanging drop technique. They belonged to the tetragonal space group P42212, with cell parameters: a = b = 103.7 Å and c = 55.7 Å, accommodating one enzyme molecule per asymmetric unit. The structure of KPR was determined by the multiwavelength anomalous dispersion method using the SeMet protein, for which data were collected to 2.3 Å resolution. The native data were collected to 1.7 Å resolution and used to refine the final structure. The secondary structure comprises 12 α-helices, three 310-helices, and 11 β-strands. The enzyme is monomeric and has two domains separated by a cleft. The N-terminal domain has an αβ-fold of the Rossmann type. The C-terminal domain (residues 170−291) is composed of eight α-helices. KPR is shown to be a member of the 6-phosphogluconate dehydrogenase C-terminal domain-like superfamily. A model for the ternary enzyme−NADPH−ketopantoate ternary complex provides a rationale for kinetic data reported for specific site-directed mutants.

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Mapping of a new pan mutation in Escherichia coli K-12

Cited by 6 publications

References 7 publications

The panE Gene, Encoding Ketopantoate Reductase, Maps at 10 Minutes and Is Allelic to apbA in Salmonella typhimurium

The panE Gene, Encoding Ketopantoate Reductase, Maps at 10 Minutes and Is Allelic to apbA in Salmonella typhimurium

PanG, a New Ketopantoate Reductase Involved in Pantothenate Synthesis

Crystal Structure of Escherichia coli Ketopantoate Reductase at 1.7 Å Resolution and Insight into the Enzyme Mechanism

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