The Isolation and Characterization of Three Types of Vitamin B6 Auxotrophs of Escherichia coli K12

Hockney, Robert C.; Scott, Thomas Allan

doi:10.1099/00221287-110-2-275

Cited by 11 publications

(11 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the absence of pyridoxal, the 'Oxidase' mutant BL-1 cannot grow and synthesizes pyridoxine at a rate about three times that of the wild-type strain (Hockney & Scott, 1979), possibly due to relief from feedback inhibition by pyridoxal 5'-phosphate. Most of this pyridoxine is excreted, and any compound that stimulates the excretion of pyridoxine probably exerts its effect by stimulating the pathway of vitamin B6 biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

“…and after 3 h the pyridoxine concentration of the medium was determined by microbiological assay (Hockney & Scott, 1979).…”

Section: Methodsmentioning

confidence: 99%

“…In the course of characterization of pdx mutations in Escherichia coli K12, we found that mutants blocked in the conversion of pyridoxine 5'-phosphate to pyridoxal 5'-phosphate (' Oxidase' mutants) could cross-feed mutants blocked at an earlier stage of vitamin B6 synthesis (Hockney & Scott, 1979). It was of interest to determine whether the excreted material responsible for the cross-feeding was pyridoxine 5'-phosphate or a derivative, possibly pyridoxine.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Synthesis of Vitamin B6 by a Mutant of Escherichia coli K12 and the Action of 4'-Deoxypyridoxine

Scott

Hockney

1979

Journal of General Microbiology

Self Cite

View full text Add to dashboard Cite

Section: Discussionmentioning

confidence: 99%

“…and after 3 h the pyridoxine concentration of the medium was determined by microbiological assay (Hockney & Scott, 1979).…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Synthesis of Vitamin B6 by a Mutant of Escherichia coli K12 and the Action of 4'-Deoxypyridoxine

Scott

Hockney

1979

Journal of General Microbiology

Self Cite

View full text Add to dashboard Cite

“…Our data show that SOR1 is necessary for synthesis of pyridoxine (vitamin B6) in C. nicotianae and in a second filamentous fungus, Aspergillus flavus. We propose that SOR1 is part of a novel pathway for de novo biosynthesis of pyridoxine distinct from the previously described Escherichia coli de novo pathway (9)(10)(11)(12)(13)(14) and that pathway divergence occurred during the evolution of the eubacteria. Finally, we also provide data demonstrating that pyridoxine quenches 1 O 2 and argue for a heretofore undiscovered role for pyridoxine in antioxidant defense.…”

mentioning

confidence: 83%

A highly conserved sequence is a novel gene involved inde novovitamin B6 biosynthesis

Ehrenshaft

Bilski

et al. 1999

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

285

278

View full text Add to dashboard Cite

The Cercospora nicotianae SOR1 (singlet oxygen resistance) gene was identified previously as a gene involved in resistance of this fungus to singlet-oxygengenerating phototoxins. Although homologues to SOR1 occur in organisms in four kingdoms and encode one of the most highly conserved proteins yet identified, the precise function of this protein has, until now, remained unknown. We show that SOR1 is essential in pyridoxine (vitamin B6) synthesis in C. nicotianae and Aspergillus flavus, although it shows no homology to previously identified pyridoxine synthesis genes identified in Escherichia coli. Sequence database analysis demonstrated that organisms encode either SOR1 or E. coli pyridoxine biosynthesis genes, but not both, suggesting that there are two divergent pathways for de novo pyridoxine biosynthesis in nature. Pathway divergence appears to have occurred during the evolution of the eubacteria. We also present data showing that pyridoxine quenches singlet oxygen at a rate comparable to that of vitamins C and E, two of the most highly efficient biological antioxidants, suggesting a previously unknown role for pyridoxine in active oxygen resistance.The filamentous, phytopathogenic fungus Cercospora nicotianae exhibits a uniquely effective, broad-spectrum resistance to potent photosensitizers of diverse chemical structure and solubility (1, 2). C. nicotianae is resistant to cercosporin, a light-activated, singlet oxygen ( 1 O 2 )-generating toxin it produces in culture and during plant parasitism, and also to other potent photosensitizers including porphyrins and xanthine and thiazine dyes. Photosensitizers are highly toxic compounds that produce their deleterious effects only after activation by light. Absorbed light energy converts the photosensitizer to an excited (triplet) state molecule that may transfer an electron to oxygen to generate superoxide and͞or transfer energy directly to oxygen, yielding 1 O 2 (3). Exposure of cells to photosensitizers plus light leads to the destruction of critical cellular components including proteins, membranes, and DNA and often results in cell death. Studies on the mechanisms by which organisms protect themselves against reactive oxygen species have focused primarily on reduced and radical forms of oxygen, including hydrogen peroxide (H 2 O 2 ), superoxide (O 2 . ), and the hydroxyl radical (OH⅐). These active oxygen species are byproducts of normal cellular metabolism, and cells contain numerous and conserved defenses against them. By contrast, the highly reactive, but nonradical 1 O 2 is produced primarily via light activation of photosensitizing compounds. Most organisms do not tolerate 1 O 2 , and few biological defenses have been identified (2). The broad-spectrum resistance expressed by Cercospora species against cercosporin and other photosensitizers of diverse structure make these organisms an excellent model for understanding the cellular basis of 1 O 2 resistance. To study specific genes and proteins involved in photosensitizer and 1 O 2 resistance...

show abstract

“…1) (10,24). However, a gene-enzyme relationship between pdxH mutations and loss of PNP/PMP oxidase was not conclusively established in these early studies, partly because of an inability to assay enzyme activity in crude extracts (11).…”

mentioning

confidence: 99%

Kinetic limitation and cellular amount of pyridoxine (pyridoxamine) 5'-phosphate oxidase of Escherichia coli K-12

1995

View full text Add to dashboard Cite

We report the purification and enzymological characterization of Escherichia coli K-12 pyridoxine (pyridoxamine) 5-phosphate (PNP/PMP) oxidase, which is a key committed enzyme in the biosynthesis of the essential coenzyme pyridoxal 5-phosphate (PLP). The enzyme encoded by pdxH was overexpressed and purified to electrophoretic homogeneity by four steps of column chromatography. The purified PdxH enzyme is a thermally stable 51-kDa homodimer containing one molecule of flavin mononucleotide (FMN). In the presence of molecular oxygen, the PdxH enzyme uses PNP or PMP as a substrate (K m ‫؍‬ 2 and 105 M and k cat ‫؍‬ 0.76 and 1.72 s ؊1 for PNP and PMP, respectively) and produces hydrogen peroxide. Thus, under aerobic conditions, the PdxH enzyme acts as a classical monofunctional flavoprotein oxidase with an extremely low k cat turnover number. Comparison of k cat /K m values suggests that PNP rather than PMP is the in vivo substrate of E. coli PdxH oxidase. In contrast, the eukaryotic enzyme has similar k cat /K m values for PNP and PMP and seems to act as a scavenger. E. coli PNP/PMP oxidase activities were competitively inhibited by the pathway end product, PLP, and by the analog, 4-deoxy-PNP, with K i values of 8 and 105 M, respectively. Immunoinhibition studies suggested that the catalytic domain of the enzyme may be composed of discontinuous residues on the polypeptide sequence. Two independent quantitation methods showed that PNP/PMP oxidase was present in about 700 to 1,200 dimer enzyme molecules per cell in E. coli growing exponentially in minimal medium plus glucose at 37؇C. Thus, E. coli PNP/PMP oxidase is an example of a relatively abundant, but catalytically sluggish, enzyme committed to PLP coenzyme biosynthesis.Pyridoxal 5Ј-phosphate (PLP) is a ubiquitous, essential coenzyme that participates in many aspects of amino acid metabolism (2). In addition, PLP is the indispensable cofactor of glycogen phosphorylases, including bacterial maltodextrin phosphorylase (19). The final steps of PLP biosynthesis start with pyridoxine (PN; vitamin B 6 ) and PN 5Ј-phosphate (PNP [ Fig. 1]), which are synthesized by many bacteria, plants, and fungi (13,23,50). In Escherichia coli K-12, PN and PNP are thought to be synthesized by a branched pathway from the committed precursors 4-phosphohydroxy-L-threonine and D-1-deoxyxylulose (13,15,22,32,33). It is not yet known whether 4-phosphohydroxy-L-threonine or its dephosphorylated derivative, 4-hydroxy-L-threonine, is condensed with D-1-deoxyxylulose to form the pyridine ring of the vitamin; the first case would give PNP and the second PN as the initial B 6 vitamer synthesized. If PN is formed directly, then the next step in the pathway is phosphorylation catalyzed by the PN/pyridoxal (PL)/pyridoxamine (PM) kinase (Fig. 1). Alternatively, if PNP is the first vitamer synthesized, then the PN/PL/PM kinase would be present in E. coli cells as part of a scavenger pathway, as it is in mammalian cells that cannot synthesize PN (28, 54).The final step in the biosynthetic pathway is ox...

show abstract

The Isolation and Characterization of Three Types of Vitamin B6 Auxotrophs of Escherichia coli K12

Cited by 11 publications

References 17 publications

Synthesis of Vitamin B6 by a Mutant of Escherichia coli K12 and the Action of 4'-Deoxypyridoxine

Synthesis of Vitamin B6 by a Mutant of Escherichia coli K12 and the Action of 4'-Deoxypyridoxine

A highly conserved sequence is a novel gene involved inde novovitamin B6 biosynthesis

Kinetic limitation and cellular amount of pyridoxine (pyridoxamine) 5'-phosphate oxidase of Escherichia coli K-12

Contact Info

Product

Resources

About