Mechanism of pyridoxine 5'-phosphate accumulation in PLPBP protein-deficiency

2022 ◽  
Author(s):  
Tomokazu Ito ◽  
Honoka Ogawa ◽  
Hisashi Hemmi ◽  
Diana M. Downs ◽  
Tohru Yoshimura
Keyword(s):  
De Novo ◽  
Binding Protein ◽  
Vitamin B ◽  
Wild Type ◽  
Salvage Pathway ◽  
Intracellular Accumulation ◽  
Pyridoxal Kinase ◽  
Genetic Studies ◽  
E Coli ◽  
Metabolic Systems

The pyridoxal 5'-phosphate (PLP)-binding protein (PLPBP) plays an important role in vitamin B 6 homeostasis. Loss of this protein in organisms such as Escherichia coli and humans disrupts the vitamin B 6 pool and induces intracellular accumulation of pyridoxine 5'-phosphate (PNP), which is normally undetectable in wild-type cells. The accumulated PNP could affect diverse metabolic systems through inhibition of some PLP-dependent enzymes. In this study, we investigated the as yet unclear mechanism of intracellular accumulation of PNP by the loss of PLPBP protein encoded by yggS in E. coli . Genetic studies using several PLPBP-deficient strains of E. coli lacking known enzyme(s) in the de novo or salvage pathway of vitamin B 6 , which includes pyridoxine (amine) 5'-phosphate oxidase (PNPO), PNP synthase, pyridoxal kinase, and pyridoxal reductase, demonstrated that neither the flux from the de novo pathway nor the salvage pathway solely contributed to the PNP accumulation caused by the PLPBP mutation. Studies with the strains lacking both PLPBP and PNPO suggested that PNP shares the same pool with PMP, and showed that PNP levels are impacted by PMP levels and vice versa . We show that disruption of PLPBP lead to perturb PMP homeostasis, which may result in PNP accumulation in the PLPBP-deficient strains. Importance A PLP-binding protein PLPBP from the conserved COG0325 family has recently been recognized as a key player in vitamin B 6 homeostasis in various organisms. Loss of PLPBP disrupts vitamin B 6 homeostasis and perturbs diverse metabolisms, including amino acid and α-keto acid metabolism. Accumulation of PNP is a characteristic phenotype of the PLPBP deficiency and is suggested to be a potential cause of the pleiotropic effects, but the mechanism of the PNP accumulation was poorly understood. In this study, we show that fluxes for PNP synthesis/metabolism are not responsible for the accumulation of PNP. Our results indicate that PLPBP is involved in the homeostasis of pyridoxamine 5'-phosphate, and its disruption may lead to the accumulation of PNP in PLPBP-deficiency.

scholarly journals Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12

1998 ◽  
Vol 180 (7) ◽  
pp. 1814-1821 ◽  
Author(s):  
Yong Yang ◽  
Ho-Ching Tiffany Tsui ◽  
Tsz-Kwong Man ◽  
Malcolm E. Winkler
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Specific Activity ◽  
Salvage Pathway ◽  
Pyridoxal Kinase ◽  
Triple Mutant ◽  
Reading Frame ◽  
E Coli ◽  
Specific Activities ◽  
K 12

ABSTRACT pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY + overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette intopdxY and crossed the resultingpdxY::ΩKanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts ofpdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show thatpdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon.pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in thetyrS-pdxY intercistronic region.

Evolution of the phenomenon of drug-resistance. II. Studies of the extracellular and intracellular derivatives of folic acid in relation to resistance to sulphathiazole and growth factor requirements

1960 ◽  
Vol 153 (951) ◽  
pp. 205-219 ◽  
Keyword(s):  
Folic Acid ◽  
Growth Factor ◽  
Resistant Strain ◽  
Sensitive Strain ◽  
Vitamin B ◽  
Intracellular Accumulation ◽  
E Coli ◽  
Dependent Strain ◽  
Resistant Strains ◽  
Derivatives Of

This study is a continuation of the results published previously (Sevag & Ishii 1958). It surveys quantitatively the extracellular and intracellular accumulation of p -aminobenzoic acid (PAB), pteridine, folic acid (FA) and citrovorum factor (CF) of the various sulphathiazole (ST)-sensitive and ST-resistant strains of Escherichia coli grown with and without ST. The altered enzymic activities of the resistant strain with respect to growth factor requirement is also determined. The following observations are made. The utilization of the exogenous PAB by the PAB-dependent strain is followed by the multiplication of cells. These events are followed by the extracellular accumulation of FA first and then CF. This pattern applies to other strains of E. coli and is in accordance with the well-known sequence of steps involved in the synthesis of PAB, pteridine, FA, CF and growth. It is shown that PAB accumulates principally extracellularly, and pteridine principally intracellularly. The synthesis of FA by the resistant strain is at least tenfold more resistant to ST than in the sensitive strain. In the resistant strain there is a greater intracellular than extracellular accumulation of FA and CF. In the sensitive strain this relationship is reversed. The resistant strains are inheritably capable of synthesizing greater amounts of pteridine, FA and CF. The PAB-dependent ST-sensitive strain can utilize a combination of 1-methionine and any of the purines, of 1-methionine alone, or vitamin B 12 alone in place of PAB for a partial or full growth. The related resistant strain, on the other hand, is unable to multiply in the salts-glucose medium with and without PAB. It requires a combination of 1-methionine and glycine for growth which cannot be replaced by any of the factors mentioned above. This requirement of the resistant strain for growth is analyzed as a deficiency of the enzymic transmethylations and transhydroxymethylation involving the function of CF in the resistant strain.

scholarly journals Transposon Insertion in the purL Gene Induces Biofilm Depletion in Escherichia coli ATCC 25922

Pathogens ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 774
Author(s):  
Virginio Cepas ◽  
Victoria Ballén ◽  
Yaiza Gabasa ◽  
Miriam Ramírez ◽  
Yuly López ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
De Novo ◽  
Wild Type ◽  
Planktonic Cells ◽  
E Coli ◽  
Qrt Pcr ◽  
Transposon Insertion ◽  
De Novo Purine Biosynthesis

Current Escherichia coli antibiofilm treatments comprise a combination of antibiotics commonly used against planktonic cells, leading to treatment failure. A better understanding of the genes involved in biofilm formation could facilitate the development of efficient and specific new antibiofilm treatments. A total of 2578 E. coli mutants were generated by transposon insertion, of which 536 were analysed in this study. After sequencing, Tn263 mutant, classified as low biofilm-former (LF) compared to the wild-type (wt) strain (ATCC 25922), showed an interruption in the purL gene, involved in the de novo purine biosynthesis pathway. To elucidate the role of purL in biofilm formation, a knockout was generated showing reduced production of curli fibres, leading to an impaired biofilm formation. These conditions were restored by complementation of the strain or addition of exogenous inosine. Proteomic and transcriptional analyses were performed to characterise the differences caused by purL alterations. Thirteen proteins were altered compared to wt. The corresponding genes were analysed by qRT-PCR not only in the Tn263 and wt, but also in clinical strains with different biofilm activity. Overall, this study suggests that purL is essential for biofilm formation in E. coli and can be considered as a potential antibiofilm target.

scholarly journals Conserved Pyridoxal 5'-Phosphate-Binding Protein YggS Impacts Amino Acid Metabolism through Pyridoxine 5'-Phosphate inEscherichia coli

2019 ◽  
Vol 85 (11) ◽  
Author(s):  
Tomokazu Ito ◽  
Kana Yamamoto ◽  
Ran Hori ◽  
Ayako Yamauchi ◽  
Diana M. Downs ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Metabolism ◽  
Biosynthetic Pathway ◽  
Acid Metabolism ◽  
Binding Protein ◽  
Protein Family ◽  
Pleiotropic Effects ◽  
Vitamin B ◽  
Content Type ◽  
E Coli

ABSTRACTEscherichia coliYggS (COG0325) is a member of the highly conserved pyridoxal 5′-phosphate (PLP)-binding protein (PLPBP) family. Recent studies suggested a role for this protein family in the homeostasis of vitamin B6and amino acids. The deletion or mutation of a member of this protein family causes pleiotropic effects in many organisms and is causative of vitamin B6-dependent epilepsy in humans. To date, little has been known about the mechanism by which lack of YggS results in these diverse phenotypes. In this study, we determined that the pyridoxine (PN) sensitivity observed inyggS-deficientE. coliwas caused by the pyridoxine 5′-phosphate (PNP)-dependent overproduction of Val, which is toxic toE. coli. The data suggest that theyggSmutation impacts Val accumulation by perturbing the biosynthetic of Thr from homoserine (Hse). Exogenous Hse inhibited the growth of theyggSmutant, caused further accumulation of PNP, and increased the levels of some intermediates in the Thr-Ile-Val metabolic pathways. Blocking the Thr biosynthetic pathway or decreasing the intracellular PNP levels abolished the perturbations of amino acid metabolism caused by the exogenous PN and Hse. Our data showed that a high concentration of intracellular PNP is the root cause of at least some of the pleiotropic phenotypes described for ayggSmutant ofE. coli.IMPORTANCERecent studies showed that deletion or mutation of members of the YggS protein family causes pleiotropic effects in many organisms. Little is known about the causes, mechanisms, and consequences of these diverse phenotypes. It was previously shown thatyggSmutations inE. coliresult in the accumulation of PNP and some metabolites in the Ile/Val biosynthetic pathway. This work revealed that some exogenous stresses increase the aberrant accumulation of PNP in theyggSmutant. In addition, the current report provides evidence indicating that some, but not all, of the phenotypes of theyggSmutant inE. coliare due to the elevated PNP level. These results will contribute to continuing efforts to determine the molecular functions of the members of the YggS protein family.

scholarly journals ExbBD-Dependent Transport of Maltodextrins through the Novel MalA Protein across the Outer Membrane of Caulobacter crescentus

2005 ◽  
Vol 187 (24) ◽  
pp. 8300-8311 ◽  
Author(s):  
Heidi Neugebauer ◽  
Christina Herrmann ◽  
Winfried Kammer ◽  
Gerold Schwarz ◽  
Alfred Nordheim ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Caulobacter Crescentus ◽  
Vitamin B ◽  
Wild Type ◽  
E Coli ◽  
Iron Source ◽  
Maltose Transport ◽  
Dependent Transport

ABSTRACT Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe3+ and vitamin B12—the substrates hitherto known to be transported by TonB-dependent transporters—the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [14C]maltodextrins from [14C]maltose to [14C]maltopentaose. [14C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a Kd of 0.2 μM, while the second transport had a Kd of 5 μM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 μM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [14C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe3+-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe3+-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with Kd values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B12 and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B12 has been demonstrated.

scholarly journals Inhibiting Pyridoxal Kinase of Entamoeba histolytica Is Lethal for This Pathogen

2021 ◽  
Vol 11 ◽  
Author(s):  
Suneeta Devi ◽  
Priya Tomar ◽  
Khaja Faisal Tarique ◽  
Samudrala Gourinath
Keyword(s):  
Entamoeba Histolytica ◽  
Drug Targets ◽  
Cultured Cells ◽  
De Novo ◽  
Active Form ◽  
Protozoan Parasite ◽  
Vitamin B ◽  
Pyridoxal Kinase ◽  
Small Molecule Libraries

Pyridoxal 5’-phosphate (PLP) functions as a cofactor for hundreds of different enzymes that are crucial to the survival of microorganisms. PLP-dependent enzymes have been extensively characterized and proposed as drug targets in Entamoeba histolytica. This pathogen is unable to synthesize vitamin B6via de-novo pathway and relies on the uptake of vitamin B6 vitamers from the host which are then phosphorylated by the enzyme pyridoxal kinase to produce PLP, the active form of vitamin B6. Previous studies from our lab shows that EhPLK is essential for the survival and growth of this protozoan parasite and its active site differs significantly with respect to its human homologue making it a potential drug target. In-silico screening of EhPLK against small molecule libraries were performed and top five ranked molecules were shortlisted on the basis of docking scores. These compounds dock into the PLP binding site of the enzyme such that binding of these compounds hinders the binding of substrate. Of these five compounds, two compounds showed inhibitory activity with IC50 values between 100-250 μM when tested in-vitro. The effect of these compounds proved to be extremely lethal for Entamoeba trophozoites in cultured cells as the growth was hampered by 91.5% and 89.5% when grown in the presence of these compounds over the period of 72 hours.

scholarly journals Successful data recovery from oscillation photographs containing strong polycrystalline diffraction rings from an unknown small-molecule contaminant: preliminary structure solution ofSalmonella typhimuriumpyridoxal kinase (PdxK)

2014 ◽  
Vol 70 (4) ◽  
pp. 526-529 ◽  
Author(s):  
G. Deka ◽  
J. N. Kalyani ◽  
J. F. Benazir ◽  
H. S. Savithri ◽  
M. R. N. Murthy
Keyword(s):  
Small Molecule ◽  
Vitamin B ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Pyridoxal Kinase ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
E Coli ◽  
Structure Solution

Pyridoxal kinase (PdxK; EC 2.7.1.35) belongs to the phosphotransferase family of enzymes and catalyzes the conversion of the three active forms of vitamin B6, pyridoxine, pyridoxal and pyridoxamine, to their phosphorylated forms and thereby plays a key role in pyridoxal 5′-phosphate salvage. In the present study, pyridoxal kinase fromSalmonella typhimuriumwas cloned and overexpressed inEscherichia coli, purified using Ni–NTA affinity chromatography and crystallized. X-ray diffraction data were collected to 2.6 Å resolution at 100 K. The crystal belonged to the primitive orthorhombic space groupP212121, with unit-cell parametersa= 65.11,b= 72.89,c= 107.52 Å. The data quality obtained by routine processing was poor owing to the presence of strong diffraction rings caused by a polycrystalline material of an unknown small molecule in all oscillation images. Excluding the reflections close to powder/polycrystalline rings provided data of sufficient quality for structure determination. A preliminary structure solution has been obtained by molecular replacement with thePhaserprogram in theCCP4 suite usingE. colipyridoxal kinase (PDB entry 2ddm) as the phasing model. Further refinement and analysis of the structure are likely to provide valuable insights into catalysis by pyridoxal kinases.

scholarly journals Thermotoga lettingae Can Salvage Cobinamide To Synthesize Vitamin B12

2013 ◽  
Vol 79 (22) ◽  
pp. 7006-7012 ◽  
Author(s):  
Nicholas C. Butzin ◽  
Michael A. Secinaro ◽  
Kristen S. Swithers ◽  
J. Peter Gogarten ◽  
Kenneth M. Noll
Keyword(s):  
Gene Cluster ◽  
Common Ancestor ◽  
De Novo ◽  
Recent Common Ancestor ◽  
Vitamin B ◽  
Ancestral State ◽  
Salvage Pathway ◽  
Content Type ◽  
Most Recent Common Ancestor

ABSTRACTWe recently reported that theThermotogalesacquired the ability to synthesize vitamin B12by acquisition of genes from two distantly related lineages,ArchaeaandFirmicutes(K. S. Swithers et al., Genome Biol. Evol. 4:730–739, 2012). Ancestral state reconstruction suggested that the cobinamide salvage gene cluster was present in theThermotogales' most recent common ancestor. We also predicted thatThermotoga lettingaecould not synthesize B12de novobut could use the cobinamide salvage pathway to synthesize B12. In this study, these hypotheses were tested, and we found thatTt. lettingaedid not synthesize B12de novobut salvaged cobinamide. The growth rate ofTt. lettingaeincreased with the addition of B12or cobinamide to its medium. It synthesized B12when the medium was supplemented with cobinamide, and no B12was detected in cells grown on cobinamide-deficient medium. Upstream of the cobinamide salvage genes is a putative B12riboswitch. In other organisms, B12riboswitches allow for higher transcriptional activity in the absence of B12. WhenTt. lettingaewas grown with no B12, the salvage genes were upregulated compared to cells grown with B12or cobinamide. Another gene cluster with a putative B12riboswitch upstream is thebtuFCDABC transporter, and it showed a transcription pattern similar to that of the cobinamide salvage genes. The BtuF proteins from species that can and cannot salvage cobinamides were shownin vitroto bind both B12and cobinamide. These results suggest thatThermotogalesspecies can use the BtuFCD transporter to import both B12and cobinamide, even if they cannot salvage cobinamide.

Bifunctional cytosolic UDP-glucose 4-epimerases catalyse the interconversion between UDP-D-xylose and UDP-L-arabinose in plants

2009 ◽  
Vol 424 (2) ◽  
pp. 169-177 ◽  
Author(s):  
Toshihisa Kotake ◽  
Ryohei Takata ◽  
Rajeev Verma ◽  
Masato Takaba ◽  
Daisuke Yamaguchi ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
De Novo ◽  
Salvage Pathway ◽  
Cell Wall Polysaccharides ◽  
Terminal Amino Acid ◽  
E Coli ◽  
Pisum Sativum L ◽  
Net Flux ◽  
Terminal Amino ◽  
Apparent Equilibrium

UDP-sugars serve as substrates in the synthesis of cell wall polysaccharides and are themselves generated through sequential interconversion reactions from UDP-Glc (UDP-glucose) as the starting substrate in the cytosol and the Golgi apparatus. For the present study, a soluble enzyme with UDP-Xyl (UDP-xylose) 4-epimerase activity was purified approx. 300-fold from pea (Pisum sativum L.) sprouts by conventional chromatography. The N-terminal amino acid sequence of the enzyme revealed that it is encoded by a predicted UDP-Glc 4-epimerase gene, PsUGE1, and is distinct from the UDP-Xyl 4-epimerase localized in the Golgi apparatus. rPsUGE1 (recombinant P. sativum UGE1) expressed in Escherichia coli exhibited both UDP-Xyl 4-epimerase and UDP-Glc 4-epimerase activities with apparent Km values of 0.31, 0.29, 0.16 and 0.15 mM for UDP-Glc, UDP-Gal (UDP-galactose), UDP-Ara (UDP-L-arabinose) and UDP-Xyl respectively. The apparent equilibrium constant for UDP-Ara formation from UDP-Xyl was 0.89, whereas that for UDP-Gal formation from UDP-Glc was 0.24. Phylogenetic analysis revealed that PsUGE1 forms a group with Arabidopsis UDP-Glc 4-epimerases, AtUGE1 and AtUGE3, apart from a group including AtUGE2, AtUGE4 and AtUGE5. Similar to rPsUGE1, recombinant AtUGE1 and AtUGE3 expressed in E. coli showed high UDP-Xyl 4-epimerase activity in addition to their UDP-Glc 4-epimerase activity. Our results suggest that PsUGE1 and its close homologues catalyse the interconversion between UDP-Xyl and UDP-Ara as the last step in the cytosolic de novo pathway for UDP-Ara generation. Alternatively, the net flux of metabolites may be from UDP-Ara to UDP-Xyl as part of the salvage pathway for Ara.

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