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.

scholarly journals Properties of human testis-specific lactate dehydrogenase expressed from Escherichia coli

1991 ◽  
Vol 273 (3) ◽  
pp. 587-592 ◽  
Author(s):  
K M LeVan ◽  
E Goldberg
Keyword(s):  
Escherichia Coli ◽  
Lactate Dehydrogenase ◽  
Prokaryotic Expression ◽  
Specific Activity ◽  
Human Testis ◽  
Reading Frame ◽  
E Coli ◽  
Heat Stable ◽  
Prokaryotic Expression Vector ◽  
Tac Promoter

The cDNA encoding the C4 isoenzyme of lactate dehydrogenase (LDH-C4) was engineered for expression in Escherichia coli. The Ldh-c open reading frame was constructed as a cassette for production of the native protein. The modified Ldh-c cDNA was subcloned into the prokaryotic expression vector pKK223-3. Transformed E. coli cells were grown to mid-exponential phase, and induced with isopropyl beta-D-thiogalactopyranoside for positive regulation of the tac promoter. Induced cells expressed the 35 kDa subunit, which spontaneously formed the enzymically active 140 kDa tetramer. Human LDH-C4 was purified over 200-fold from litre cultures of cells by AMP and oxamate affinity chromatography to a specific activity of 106 units/mg. The enzyme was inhibited by pyruvate concentrations above 0.3 mM, had a Km for pyruvate of 0.03 mM, a turnover number (nmol of NADH oxidized/mol of LDH-C4 per min at 25 degrees C) of 14,000 and was heat-stable.

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 A Tetrahydrofolate-Dependent O-Demethylase, LigM, Is Crucial for Catabolism of Vanillate and Syringate in Sphingomonas paucimobilis SYK-6

2005 ◽  
Vol 187 (6) ◽  
pp. 2030-2037 ◽  
Author(s):  
Tomokuni Abe ◽  
Eiji Masai ◽  
Keisuke Miyauchi ◽  
Yoshihiro Katayama ◽  
Masao Fukuda
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Double Mutant ◽  
Open Reading Frame ◽  
Sphingomonas Paucimobilis ◽  
Reading Frame ◽  
E Coli ◽  
Demethylase Activity ◽  
Specific Activities

ABSTRACT Vanillate and syringate are converted into protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively, by O-demethylases in Sphingomonas paucimobilis SYK-6. PCA is further degraded via the PCA 4,5-cleavage pathway, while 3MGA is degraded through multiple pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and an unidentified 3MGA O-demethylase and gallate dioxygenase are participants. For this study, we isolated a 4.7-kb SmaI fragment that conferred on Escherichia coli the activity required for the conversion of vanillate to PCA. The nucleotide sequence of this fragment revealed an open reading frame of 1,413 bp (ligM), the deduced amino acid sequence of which showed 49% identity with that of the tetrahydrofolate (H4folate)-dependent syringate O-demethylase gene (desA). The metF and ligH genes, which are thought to be involved in H4folate-mediated C1 metabolism, were located just downstream of ligM. The crude LigM enzyme expressed in E. coli converted vanillate and 3MGA to PCA and gallate, respectively, with similar specific activities, and only in the presence of H4folate; however, syringate was not a substrate for LigM. The disruption of ligM led to significant growth retardation on both vanillate and syringate, indicating that ligM is involved in the catabolism of these substrates. The ability of the ligM mutant to transform vanillate was markedly decreased, and this mutant completely lost the 3MGA O-demethylase activity. A ligM desA double mutant completely lost the ability to transform vanillate, thus indicating that desA also contributes to vanillate degradation. All of these results indicate that ligM encodes vanillate/3MGA O-demethylase and plays an important role in the O demethylation of vanillate and 3MGA, respectively.

Cloning and expression of the beta-D-phosphogalactoside galactohydrolase gene of Lactobacillus casei in Escherichia coli K-12

1982 ◽  
Vol 152 (3) ◽  
pp. 1138-1146
Author(s):  
L J Lee ◽  
J B Hansen ◽  
E K Jagusztyn-Krynicka ◽  
B M Chassy
Keyword(s):  
Escherichia Coli ◽  
Lactobacillus Casei ◽  
Specific Activity ◽  
Protein Product ◽  
Coli Strain ◽  
Cloning And Expression ◽  
E Coli ◽  
Lactose Metabolism ◽  
Gene Coding ◽  
K 12

Lactose metabolism in Lactobacillus casei 64H is associated with the presence of plasmid pLZ64. This plasmid determines both phosphoenolpyruvate-dependent phosphotransferase uptake of lactose and beta-D-phosphogalactoside galactohydrolase. A shotgun clone bank of chimeric plasmids containing restriction enzyme digest fragments of pLZ64 DNA was constructed in Escherichia coli K-12. One clone contained the gene coding for beta-D-phosphogalactoside galactohydrolase on a 7.9-kilobase PstI fragment cloned into the vector pBR322 in E. coli strain chi 1849. The beta-D-phosphogalactoside galactohydrolase enzyme isolated from E. coli showed no difference from that isolated from L. casei, and specific activity of beta-D-phosphogalactoside galactohydrolase was stimulated 1.8-fold in E. coli by growth in media containing beta-galactosides. A restriction map of the recombinant plasmid was compiled, and with that information, a series of subclones was constructed. From an analysis of the proteins produced by minicells prepared from transformant E. coli cells containing each of the recombinant subclone plasmids, it was found that the gene for the 56-kilodalton beta-D-phosphogalactoside galactohydrolase was transcribed from an L. casei-derived promoter. The gene for a second protein product (43 kilodaltons) was transcribed in the opposite direction, presumably under the control of a promoter in pBR322. The relationship of this second product to the lactose metabolism genes of L. casei is at present unknown.

scholarly journals Construction of Deoxyriboaldolase-Overexpressing Escherichia coli and Its Application to 2-Deoxyribose 5-Phosphate Synthesis from Glucose and Acetaldehyde for 2′-Deoxyribonucleoside Production

2003 ◽  
Vol 69 (7) ◽  
pp. 3791-3797 ◽  
Author(s):  
Nobuyuki Horinouchi ◽  
Jun Ogawa ◽  
Takafumi Sakai ◽  
Takako Kawano ◽  
Seiichiro Matsumoto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Specific Activity ◽  
Cell Extract ◽  
Predicted Amino Acid Sequence ◽  
Enzymatic Reactions ◽  
Chromosomal Dna ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli

ABSTRACT The gene encoding a deoxyriboaldolase (DERA) was cloned from the chromosomal DNA of Klebsiella pneumoniae B-4-4. This gene contains an open reading frame consisting of 780 nucleotides encoding 259 amino acid residues. The predicted amino acid sequence exhibited 94.6% homology with the sequence of DERA from Escherichia coli. The DERA of K. pneumoniae was expressed in recombinant E. coli cells, and the specific activity of the enzyme in the cell extract was as high as 2.5 U/mg, which was threefold higher than the specific activity in the K. pneumoniae cell extract. One of the E. coli transformants, 10B5/pTS8, which had a defect in alkaline phosphatase activity, was a good catalyst for 2-deoxyribose 5-phosphate (DR5P) synthesis from glyceraldehyde 3-phosphate and acetaldehyde. The E. coli cells produced DR5P from glucose and acetaldehyde in the presence of ATP. Under the optimal conditions, 100 mM DR5P was produced from 900 mM glucose, 200 mM acetaldehyde, and 100 mM ATP by the E. coli cells. The DR5P produced was further transformed to 2′-deoxyribonucleoside through coupling the enzymatic reactions of phosphopentomutase and nucleoside phosphorylase. These results indicated that production of 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase is possible with the addition of a suitable energy source, such as ATP.

Purification and characterization of D-β-hydroxybutyrate dehydrogenase expressed in Escherichia coli

1993 ◽  
Vol 71 (7-8) ◽  
pp. 406-410
Author(s):  
Les Jones ◽  
Sharon Churchill ◽  
Perry Churchill
Keyword(s):  
Escherichia Coli ◽  
Kinetic Parameters ◽  
Functional Properties ◽  
Rat Liver ◽  
Specific Activity ◽  
Purification And Characterization ◽  
E Coli ◽  
Specific Activities ◽  
Viable Method

D-β-Hydroxybutyrate dehydrogenase (BDH), a lipid-requiring enzyme, has been cloned into pUC18, expressed in Escherichia coli, and purified to homogeneity. The apoenzyme, i.e., the enzyme devoid of phospholipid, has no activity, but can be activated by phospholipid to a specific activity of 129 μmol/(min∙mg). The functional properties of the enzyme expressed in E. coli were compared with the enzyme purified from rat liver. The specific activities, kinetic parameters, and phospholipid activation profiles were virtually identical. These results indicate that the expression of the enzyme in E. coli is a viable method for producing active functional BDH and should allow for the production of specifically altered BDH molecules.Key words: D-β-hydroxybutyrate dehydrogenase, cloning, expression, lipid requiring.

Identification of a trans-dominant mutation affecting proline dehydrogenase in Escherichia coli

1985 ◽  
Vol 31 (11) ◽  
pp. 988-993 ◽  
Author(s):  
Charles E. Deutch ◽  
John M. O'Brien Jr. ◽  
Michael S. VanNieuwenhze
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Parent Strain ◽  
Specific Activity ◽  
Deficient Mutant ◽  
Proline Dehydrogenase ◽  
Dominant Mutation ◽  
E Coli ◽  
F Factor ◽  
K 12

L-Proline dehydrogenase catalyzes the oxidation of L-proline to Δ1-pyrroline-5-carboxylate, a reaction that is an important step in the utilization of proline as a carbon or nitrogen source by bacteria. A mutant of Escherichia coli K-12 lacking L-leucyl-tRNA:protein transferase had been found previously to contain about five times as much proline dehydrogenase activity as its parent strain. This difference has now been shown to be due to the presence in the parent strain of a previously unrecognized mutation. This mutation, which has been designated put-4977, specifically affects proline dehydrogenase rather than proline uptake. Although proline dehydrogenase remains inducible by L-proline in strains carrying the mutation, there is a premature cessation of differential synthesis during induction that results in a lower specific activity. The mutation shows about 50% P1-mediated cotransduction with pyrC and is therefore located at about 22 min on the E. coli chromosome. Merodiploids containing a normal F′ factor still exhibit decreased enzyme activity, indicating that the put-4977 mutation is trans-dominant. The mutation cannot be detected in present stocks of the transferase-deficient mutant, suggesting that this mutant is a revertant for put-4977.

scholarly journals Crystal Structure of Pyridoxal Kinase from the Escherichia coli pdxK Gene: Implications for the Classification of Pyridoxal Kinases

2006 ◽  
Vol 188 (12) ◽  
pp. 4542-4552 ◽  
Author(s):  
Martin K. Safo ◽  
Faik N. Musayev ◽  
Martino L. di Salvo ◽  
Sharyn Hunt ◽  
Jean-Baptiste Claude ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Active Site ◽  
Pyridoxal Phosphate ◽  
Salvage Pathway ◽  
Pyridoxal Kinase ◽  
E Coli ◽  
Binary Complexes ◽  
Significant Conformational Change

ABSTRACT The pdxK and pdxY genes have been found to code for pyridoxal kinases, enzymes involved in the pyridoxal phosphate salvage pathway. Two pyridoxal kinase structures have recently been published, including Escherichia coli pyridoxal kinase 2 (ePL kinase 2) and sheep pyridoxal kinase, products of the pdxY and pdxK genes, respectively. We now report the crystal structure of E. coli pyridoxal kinase 1 (ePL kinase 1), encoded by a pdxK gene, and an isoform of ePL kinase 2. The structures were determined in the unliganded and binary complexes with either MgATP or pyridoxal to 2.1-, 2.6-, and 3.2-Å resolutions, respectively. The active site of ePL kinase 1 does not show significant conformational change upon binding of either pyridoxal or MgATP. Like sheep PL kinase, ePL kinase 1 exhibits a sequential random mechanism. Unlike sheep pyridoxal kinase, ePL kinase 1 may not tolerate wide variation in the size and chemical nature of the 4′ substituent on the substrate. This is the result of differences in a key residue at position 59 on a loop (loop II) that partially forms the active site. Residue 59, which is His in ePL kinase 1, interacts with the formyl group at C-4′ of pyridoxal and may also determine if residues from another loop (loop I) can fill the active site in the absence of the substrate. Both loop I and loop II are suggested to play significant roles in the functions of PL kinases.

scholarly journals Rhs Elements Comprise Three Subfamilies Which Diverged Prior to Acquisition by Escherichia coli

1998 ◽  
Vol 180 (16) ◽  
pp. 4102-4110 ◽  
Author(s):  
Yong-Dong Wang ◽  
Sheng Zhao ◽  
Charles W. Hill
Keyword(s):  
Escherichia Coli ◽  
Phylogenetic Analysis ◽  
Open Reading Frame ◽  
Large Protein ◽  
Rich Region ◽  
Reading Frame ◽  
E Coli ◽  
The Core ◽  
Reference Collection ◽  
K 12

ABSTRACT The Rhs elements are complex genetic composites widely spread among Escherichia coli isolates. One of their components, a 3.7-kb, GC-rich core, maintains a single open reading frame that extends the full length of the core and then 400 to 600 bp beyond into an AT-rich region. Whereas Rhs cores are homologous, core extensions from different elements are dissimilar. Two new Rhs elements from strains of the ECOR reference collection have been characterized. RhsG (from strain ECOR-11) maps to min 5.3, and RhsH (from strain ECOR-45) maps to min 32.8, where it lies in tandem with RhsE. Comparison of strain K-12 to ECOR-11 indicates that RhsGwas once present in but has been largely deleted from an ancestor of K-12. Phylogenetic analysis shows that the cores from eight known elements fall into three subfamilies, RhsA-B-C-F,RhsD-E, and RhsG-H. Cores from different subfamilies diverge 22 to 29%. Analysis of substitutions that distinguish between subfamilies shows that the origin of the ancestral core as well as the process of subfamily separation occurred in a GC-rich background. Furthermore, each subfamily independently passed from the GC-rich background to a less GC-rich background such asE. coli. A new example of core-extension shuffling provides the first example of exchange between cores of different subfamilies. A novel component of RhsE and RhsG,vgr, encodes a large protein distinguished by 18 to 19 repetitions of a Val-Gly dipeptide occurring with a eight-residue periodicity.

scholarly journals Correlation of Rhs elements with Escherichia coli population structure.

Genetics ◽  
1995 ◽  
Vol 141 (1) ◽  
pp. 15-24
Author(s):  
C W Hill ◽  
G Feulner ◽  
M S Brody ◽  
S Zhao ◽  
A B Sadosky ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Open Reading Frame ◽  
Clonal Structure ◽  
Reading Frame ◽  
E Coli ◽  
Sequence Identity ◽  
Reference Collection ◽  
K 12 ◽  
Recent Variation ◽  
Fourth Member

Abstract The Rhs family of composite genetic elements was assessed for variation among independent Escherichia coli strains of the ECOR reference collection. The location and content of the RhsA-B-C-F subfamily correlates highly with the clonal structure of the ECOR collection. This correlation exists at several levels: the presence of Rhs core homology in the strain, the location of the Rhs elements present, and the identity of the Rhs core-extensions associated with each element. A provocative finding was that an identical 1518-bp segment, covering core-extension-b1 and its associated downstream open reading frame, is present in two distinct clonal groups, but in association with different Rhs elements. The sequence identity of this segment when contrasted with the divergence of other chromosomal segments suggests that shuffling of Rhs core extensions has been a relatively recent variation. Nevertheless the copies of core-extension-b1 were placed within the respective Rhs elements before the emergence of the clonal groups. In the course of this analysis, two new Rhs elements absent from E. coli K-12 were discovered: RhsF, a fourth member of the RhsA-B-C-F subfamily, and RhsG, the prototype of a third Rhs subfamily.

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