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.

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.

High Expression of Human Amelogenin in E. Coli

1996 ◽  
Vol 10 (2) ◽  
pp. 187-194 ◽  
Author(s):  
D. Deutsch ◽  
E. Chityat ◽  
M. Hekmati ◽  
A. Palmon ◽  
Y. Farkash ◽  
...  
Keyword(s):  
Amino Acid ◽  
Western Blotting ◽  
Rt Pcr ◽  
Amino Acid Sequencing ◽  
Terminal Amino Acid ◽  
Expression Plasmid ◽  
Over Expression ◽  
E Coli ◽  
Sds Page ◽  
Terminal Amino

A human cDNA, encoding for the 175-aminoacid human amelogenin, was prepared by RT PCR from tooth bud mRNA and sub-cloned into pGEX-KG expression plasmid for over-expression in E. coli. The expressed protein was characterized by SDS-PAGE, Western blotting, and N-terminal amino acid sequencing.

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.

Kynurenine aminotransferase and glutamine transaminase K of Escherichia coli: identity with aspartate aminotransferase

2001 ◽  
Vol 360 (3) ◽  
pp. 617-623 ◽  
Author(s):  
Qian HAN ◽  
Jianmin FANG ◽  
Jianyong LI
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Aspartate Aminotransferase ◽  
Expression System ◽  
Xanthurenic Acid ◽  
Amino Acid Residues ◽  
Terminal Amino Acid ◽  
Kynurenine Aminotransferase ◽  
E Coli ◽  
Terminal Amino

The present study describes the isolation of a protein from Escherichia coli possessing kynurenine aminotransferase (KAT) activity and its identification as aspartate aminotransferase (AspAT). KAT catalyses the transamination of kynurenine and 3-hydroxykynurenine to kynurenic acid and xanthurenic acid respectively, and the enzyme activity can be easily detected in E. coli cells. Separation of the E. coli protein possessing KAT activity through various chromatographic steps led to the isolation of the enzyme. N-terminal sequencing of the purified protein determined its first 10 N-terminal amino acid residues, which were identical with those of the E. coli AspAT. Recombinant AspAT (R-AspAT), homologously expressed in an E. coli/pET22b expression system, was capable of catalysing the transamination of both l-kynurenine (Km = 3mM; Vmax = 7.9μmol·min−1·mg−1) and 3-hydroxy-dl-kynurenine (Km = 3.7mM; Vmax = 1.25μmol·min−1·mg−1) in the presence of pyruvate as an amino acceptor, and exhibited its maximum activity at temperatures between 50–60°C and at a pH of approx. 7.0. Like mammalian KATs, R-AspAT also displayed high glutamine transaminase K activity when l-phenylalanine was used as an amino donor (Km = 8mM; Vmax = 20.6μmol·min−1·mg−1). The exact match of the first ten N-terminal amino acid residues of the KAT-active protein with that of AspAT, in conjunction with the high KAT activity of R-AspAT, provides convincing evidence that the identity of the E. coli protein is AspAT.

A ribosomal-boand aminopeptidase in Escherichia coli B: substrate specificity

1970 ◽  
Vol 48 (12) ◽  
pp. 1292-1296 ◽  
Author(s):  
A. T. Matheson ◽  
A. J. Dick ◽  
F. Rollin
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Peptide Bond ◽  
Terminal Amino Acid ◽  
Strong Activity ◽  
E Coli ◽  
Rate Of Hydrolysis ◽  
Terminal Amino ◽  
Hydrolysis Of ◽  
Escherichia Coli B

The substrate specificity of the ribosomal-bound aminopeptidase from Escherichia coli B has been studied using di-, tri-, and tetrapeptides. The enzyme shows strong activity to leucyl, methionyl, threonyl, and lysyl peptides. Of the other dipeptides tested considerable hydrolysis was observed only if the C-terminal amino acid was leucine or methionine. In a given series of peptides the rate of hydrolysis of the N-terminal peptide bond increased as the size of the peptide increased. Although leucyi dipeptides were hydroiyzed more rapidly than the corresponding methionyl dipeptide the reverse was true with the tripeptides tested. No carboxypeptidase activity was observed and peptides containing D-amino acids were not hydroiyzed. The substrate specificity of the aminopeptidase was compared with the known N-terminal sequences of E. coli proteins to determine whether the enzyme may be involved in the removal of N-formylmethionyl from newly synthesized polypeptides.

scholarly journals A cyanogen bromide fragment of β-galactosidase from Escherichia coli with α-donor activity in complementation of the enzyme from mutant M15

1976 ◽  
Vol 155 (2) ◽  
pp. 209-216 ◽  
Author(s):  
D V. Marinkovic ◽  
J N. Marinkovic
Keyword(s):  
Escherichia Coli ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Terminal Amino Acid ◽  
E Coli ◽  
Molecular Weights ◽  
Terminal Amino ◽  
Cyanogen Bromide Fragment ◽  
Donor Activity

Aminoethylated β-galactosidase from Escherichia coli was cleaved by CNBr. The fragment C4a was purified by gel filtration and ion-exchange chromatography. The molecular weight of the fragment C4a was determined to be 9000 +/- 600. The N-terminal amino acid was found to be isoleucine. Qualitative examination of homogeneity was carried out by disc-gel electrophoresis. The fragment C4a was shown to be active as an α donor in complementation of β-galactosidase activity in vitro with E. coli mutant M15, which has a deletion in the α region of the z gene. The molecular weights of complementable fractions from mutant M15 were found to be 123 000 +/- 2500 and 507 000 +/- 11 000, and of the complemented enzyme 522 500 +/- 11 400.

scholarly journals β-Galactosidase II in Ripening Tomatoes

HortScience ◽  
1995 ◽  
Vol 30 (4) ◽  
pp. 817A-817
Author(s):  
Russell Pressey ◽  
C.M. Sean Carrington
Keyword(s):  
Amino Acid ◽  
Cell Walls ◽  
Amino Acid Sequences ◽  
Cell Wall Polysaccharides ◽  
Terminal Amino Acid ◽  
Molecular Weights ◽  
Sds Page ◽  
Original Procedure ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

Tomatoes contain several isozymes of β-galactosidase, but only one, β-galactosidase II, can hydrolyze the β-1,4-galactans in tomato cell walls. β-galactosidase II has now been highly purified by modification of the original procedure. The molecular weight of this isozyme is ≈62 kDa according to gel infiltration, but SDS-PAGE of the purified enzyme separated three components with molecular weights of 29, 42, and 82 kDa. The 82-kDa peptide may be the intact enzyme and the smallest peptides are subunits as proposed for other β-galactosidases. The N-terminal amino acid sequence of β-galactosidase II showed high homology with amino acid sequences reported for other plant β-galactosidases. A new assay for β-galactosidase II in tomato extracts has been developed using FPLC. This isozyme was not detected in mature-green tomatoes but appeared at about the breaker stage and increased during ripening. The increase in b-galactosidase II was accompanied by a decrease in galactose content of cell wall polysaccharides, suggesting that this enzyme may be involved in the loss of galactose during tomato ripening.

scholarly journals The N-Acetylmuramic Acid 6-Phosphate Phosphatase MupP Completes the Pseudomonas Peptidoglycan Recycling Pathway Leading to Intrinsic Fosfomycin Resistance

mBio ◽  
2017 ◽  
Vol 8 (2) ◽  
Author(s):  
Marina Borisova ◽  
Jonathan Gisin ◽  
Christoph Mayer
Keyword(s):  
Cell Wall ◽  
De Novo ◽  
Gram Negative Bacteria ◽  
Salvage Pathway ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
E Coli ◽  
A Cell ◽  
Recycling Pathway ◽  
Novel Drug

ABSTRACT Bacterial cells are encased in and stabilized by a netlike peptidoglycan (PGN) cell wall that undergoes turnover during bacterial growth. PGN turnover fragments are frequently salvaged by the cells via a pathway referred to as PGN recycling. Two different routes for the recycling of the cell wall sugar N-acetylmuramic acid (MurNAc) have been recognized in bacteria. In Escherichia coli and related enterobacteria, as well as in most Gram-positive bacteria, MurNAc is recovered via a catabolic route requiring a MurNAc 6-phosphate etherase (MurQ in E. coli) enzyme. However, many Gram-negative bacteria, including Pseudomonas species, lack a MurQ ortholog and use an alternative, anabolic recycling route that bypasses the de novo biosynthesis of uridyldiphosphate (UDP)-MurNAc, the first committed precursor of PGN. Bacteria featuring the latter pathway become intrinsically resistant to the antibiotic fosfomycin, which targets the de novo biosynthesis of UDP-MurNAc. We report here the identification and characterization of a phosphatase enzyme, named MupP, that had been predicted to complete the anabolic recycling pathway of Pseudomonas species but has remained unknown so far. It belongs to the large haloacid dehalogenase family of phosphatases and specifically converts MurNAc 6-phosphate to MurNAc. A ΔmupP mutant of Pseudomonas putida was highly susceptible to fosfomycin, accumulated large amounts of MurNAc 6-phosphate, and showed lower levels of UDP-MurNAc than wild-type cells, altogether consistent with a role for MupP in the anabolic PGN recycling route and as a determinant of intrinsic resistance to fosfomycin. IMPORTANCE Many Gram-negative bacteria, but not E. coli, make use of a cell wall salvage pathway that contributes to the pool of UDP-MurNAc, the first committed precursor of cell wall synthesis in bacteria. This salvage pathway is of particular interest because it confers intrinsic resistance to the antibiotic fosfomycin, which blocks de novo UDP-MurNAc biosynthesis. Here we identified and characterized a previously missing enzyme within the salvage pathway, the MurNAc 6-phosphate phosphatase MupP of P. putida. MupP, together with the other enzymes of the anabolic recycling pathway, AnmK, AmgK, and MurU, yields UDP-MurNAc, renders bacteria intrinsically resistant to fosfomycin, and thus may serve as a novel drug target for antimicrobial therapy. IMPORTANCE Many Gram-negative bacteria, but not E. coli, make use of a cell wall salvage pathway that contributes to the pool of UDP-MurNAc, the first committed precursor of cell wall synthesis in bacteria. This salvage pathway is of particular interest because it confers intrinsic resistance to the antibiotic fosfomycin, which blocks de novo UDP-MurNAc biosynthesis. Here we identified and characterized a previously missing enzyme within the salvage pathway, the MurNAc 6-phosphate phosphatase MupP of P. putida. MupP, together with the other enzymes of the anabolic recycling pathway, AnmK, AmgK, and MurU, yields UDP-MurNAc, renders bacteria intrinsically resistant to fosfomycin, and thus may serve as a novel drug target for antimicrobial therapy.

scholarly journals Comparison of protein patterns among some Salmonella serovars and E. coli by two-dimensional polyacrylamide gel electrophoresis

1970 ◽  
Vol 6 (2) ◽  
pp. 127-138
Author(s):  
F Begum ◽  
Y Adachi ◽  
MSR Khan
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Two Dimensional ◽  
Protein Patterns ◽  
Terminal Amino Acid ◽  
E Coli ◽  
Terminal Amino ◽  
Salmonella Serovars

The study was conducted to compare the protein patterns among some Salmonella serovars and E. coli using Two Dimensional Polyacrylamide Gel Electophoresis. The Two Dimensional Polyacrylamide Gel Electophoresis showed a 37.81 kDa well separated protein spots with all Salmonella serovars at the same time with E. coli a 36.5 kDa protein. However, these protein spots of Two Dimensional Polyacrylamide Gel Electophoresis were further tested with Immunoblotting analysis with specific antiserum against Salmonella typhimurium infected chicks. All selected Salmonella serovars successfully identified a common 37.81 kDa protein whereas E. coli spots identified as 36.5 kDa protein instead of 37.81 kDa. As a further monitoring of these proteins as to check the homogeneity and heterogeneity for N-terminal amino acid sequencing, the specific protein bands from all Salmonella serovars and E. coli were excised, purified and subjected to sequence analysis. The amino acid sequence alignment showed the 37.81 kDa proteins of some Salmonella serovars were identical or homologous among the Salmonella serovars. The N-terminal amino acid alignments of the 37.81 kDa proteins were determined as alanineglutamine- valine-isoleucine-asparagine-threonine-asparagine. On the other hand, the N-terminal amino acid alignment of the 36.5 kDa protein of E. coli ACLD2201 was found to be heterologous as alanine-proline-lysine-aspartic acid-aspararginethreonine- tryptophan. The findings of this study can be concluded that the 37.81 kDa protein of some Salmonella serovars and 36.5 kDa protein of E. coli were completely different though there is some identity of these organisms due to the presence of Enterobacterial common antigen. Key words: Salmonella, 2D-PAGE, amino acid sequence doi: 10.3329/bjvm.v6i2.2324 Bangl. J. Vet. Med. (2008). 6 (2): 127-138

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