scholarly journals 6-Phosphogluconate dehydrogenase from Lactococcus lactis: a role for arginine residues in binding substrate and coenzyme

1999 ◽  
Vol 338 (1) ◽  
pp. 55-60 ◽  
Author(s):  
Emmanuel TETAUD ◽  
Stefania HANAU ◽  
Jeremy M. WELLS ◽  
Richard W. F. Le PAGE ◽  
Margaret J. ADAMS ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Gene Encoding ◽  
E Coli ◽  
Coenzyme Binding ◽  
Phosphogluconate Dehydrogenase ◽  
Arginine Residues ◽  
Binding Pockets ◽  
Binding Residues ◽  
Cloned Gene ◽  
Binding Substrate

A gene encoding 6-phosphogluconate dehydrogenase (6-PGDH, EC 1.1.1.44) was identified from the homofermentative lactic acid bacterium Lactococcus lactis, by complementation of Escherichia coli mutants. The cloned gene was then expressed to high levels in E. coli and the protein purified for kinetic analysis. The enzyme had a Km for 6-phosphogluconate of 15.4±1.4 µM and for NADP of 1.9±0.2 µM at pH 7.5. Sequence comparison of the L. lactis 6-PGDH with the corresponding enzyme derived from the pathogenic protozoan Trypanosoma brucei and sheep liver revealed the substrate-binding residues to be identical in all three species, although the three coenzyme-binding pockets differed slightly. A totally conserved arginine residue (Arg-447), believed to bind the 6-phosphate of substrate, was mutated to lysine, aspartate, alanine or tryptophan. In each case enzyme activity was lost, confirming an essential role for this residue on activity. A second arginine (Arg-34), believed to be critical in binding the 2´-phosphate of cofactor NADP+, was mutated to a tyrosine residue, as found in one atypical isoform of the enzyme in Bacillus subtilis. This alteration led to decrease in affinity for NADP+ of nearly three orders of magnitude. A second 6-PGDH gene has been identified from the genome of B. subtilis. This second isoform contains an arginine (Arg-34) in this position, suggesting that B. subtilis has two 6-PGDHs with different coenzyme specificities.

scholarly journals 6-Phosphogluconate dehydrogenase from Lactococcus lactis: a role for arginine residues in binding substrate and coenzyme

1999 ◽  
Vol 338 (1) ◽  
pp. 55 ◽  
Author(s):  
Emmanuel TETAUD ◽  
Stefania HANAU ◽  
Jeremy M. WELLS ◽  
Richard W.F. Le PAGE ◽  
Margaret J. ADAMS ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Phosphogluconate Dehydrogenase ◽  
Arginine Residues ◽  
Binding Substrate

scholarly journals Plasmid pCS1966, a New Selection/Counterselection Tool for Lactic Acid Bacterium Strain Construction Based on the oroP Gene, Encoding an Orotate Transporter from Lactococcus lactis

2008 ◽  
Vol 74 (15) ◽  
pp. 4772-4775 ◽  
Author(s):  
Christian Solem ◽  
Els Defoor ◽  
Peter Ruhdal Jensen ◽  
Jan Martinussen
Keyword(s):  
Lactococcus Lactis ◽  
Triosephosphate Isomerase ◽  
Attachment Site ◽  
Auxotrophic Mutant ◽  
Synthetic Promoter ◽  
Gene Encoding ◽  
E Coli ◽  
Bacterium Strain ◽  
Selection For ◽  
Different Levels

ABSTRACT In this paper we describe the new selection/counterselection vector pCS1966, which is suitable for both sequence-specific integration based on homologous recombination and integration in a bacteriophage attachment site. This plasmid harbors oroP, which encodes a dedicated orotate transporter, and can replicate only in Escherichia coli. Selection for integration is performed primarily by resistance to erythromycin; alternatively, the ability to utilize orotate as a pyrimidine source in a pyrimidine auxotrophic mutant could be utilized. Besides allowing the cell to utilize orotate, the transporter renders the cell sensitive to 5-fluoroorotate. This sensitivity is used to select for loss of the plasmid. When expressed from its own promoter, oroP was toxic to E. coli, whereas in Lactococcus lactis the level of expression of oroP from a chromosomal copy was too low to confer 5-fluoroorotate sensitivity. In order to obtain a plasmid that confers 5-fluoroorotate sensitivity when it is integrated into the chromosome of L. lactis and at the same time can be stably maintained in E. coli, the expression of the oroP gene was controlled from a synthetic promoter conferring these traits. To demonstrate its use, a number of L. lactis strains expressing triosephosphate isomerase (tpiA) at different levels were constructed.

scholarly journals GENETIC ANALYSIS OF STAPHYLOCOCCAL NUCLEASE: IDENTIFICATION OF THREE INTRAGENIC "GLOBAL" SUPPRESSORS OF NUCLEASE-MINUS MUTATIONS

Genetics ◽  
1985 ◽  
Vol 110 (4) ◽  
pp. 539-555 ◽  
Author(s):  
David Shortle ◽  
Beth Lin
Keyword(s):  
Nuclease Activity ◽  
Staphylococcal Nuclease ◽  
Missense Mutations ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Protein Coding ◽  
E Coli ◽  
Suppressor Mutations ◽  
Cloned Gene

ABSTRACT A collection of 77 unique missense mutations distributed across the gene encoding staphylococcal nuclease (nuc) has been assembled. These mutations were induced by random gap misrepair mutagenesis of the cloned gene and were identified in E. coli transformants expressing reduced levels of nuclease activity. Four nuc  - mutations which alter amino acid residues at positions outside of the active site region of the enzyme were submitted to a second round of mutagenesis, and characterization of several independent NUC+ isolates lead to the identification of three second-site suppressor mutations within the protein-coding sequence of the nuc gene. On separation from the mutation originally suppressed and recombination with a number of other nuc  - mutations, all three suppressors displayed the property of "global" suppression, i.e., phenotypic suppression of the nuclease-minus character of multiple different alleles. A simple and generally applicable strategy was used to obtain efficient homologous recombination between plasmids for purposes of mapping nuc  - mutations, mapping second-site suppressors and constructing double mutant combinations from pairs of single mutations.

scholarly journals Acquisition of the rfb-gnd Cluster in Evolution of Escherichia coli O55 and O157

2000 ◽  
Vol 182 (21) ◽  
pp. 6183-6191 ◽  
Author(s):  
Phillip I. Tarr ◽  
Laura M. Schoening ◽  
Yoo-Lee Yea ◽  
Teresa R. Ward ◽  
Srdjan Jelacic ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Side Chains ◽  
Gene Encoding ◽  
Gram Negative ◽  
E Coli ◽  
Phosphogluconate Dehydrogenase ◽  
Serovar Typhimurium ◽  
Polymorphic Gene ◽  
O Antigens

ABSTRACT The rfb region specifies the structure of lipopolysaccharide side chains that comprise the diverse gram-negative bacterial somatic (O) antigens. The rfb locus is adjacent to gnd, which is a polymorphic gene encoding 6-phosphogluconate dehydrogenase. To determine if rfb andgnd cotransfer, we sequenced gnd in five O55 and 13 O157 strains of Escherichia coli. E. coli O157:H7 has a gnd allele (allele A) that is only 82% identical to the gnd allele (alleleD) of closely related E. coli O55:H7. In contrast, gnd alleles of E. coli O55 in distant lineages are >99.9% identical to gnd alleleD. Though gnd alleles B andC in E. coli O157 that are distantly related toE. coli O157:H7 are more similar to allele Athan to allele D, there are nucleotide differences at 4 to 6% of their sites. Alleles B and C can be found in E. coli O157 in different lineages, but we have found allele A only in E. coli O157 belonging to the DEC5 lineage. DNA 3′ to the O55 gnd allele in diverse E. coli lineages has sequences homologous totnpA of the Salmonella enterica serovar Typhimurium IS200 element, E. coli Rhs elements (including an H-rpt gene), and portions of the O111 and O157rfb regions. We conclude that rfb andgnd cotransferred into E. coli O55 and O157 in widely separated lineages and that recombination was responsible for recent antigenic shifts in the emergence of pathogenic E. coli O55 and O157.

scholarly journals The cloning and sequence analysis of the aspC and tyrB genes from Escherichia coli K12. Comparison of the primary structures of the aspartate aminotransferase and aromatic aminotransferase of E. coli with those of the pig aspartate aminotransferase isoenzymes

1986 ◽  
Vol 234 (3) ◽  
pp. 593-604 ◽  
Author(s):  
I G Fotheringham ◽  
S A Dacey ◽  
P P Taylor ◽  
T J Smith ◽  
M G Hunter ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
Aspartate Aminotransferase ◽  
Gene Encoding ◽  
E Coli ◽  
Escherichia Coli K12 ◽  
Coenzyme Binding ◽  
Catalytic Mechanisms ◽  
Substrate Binding Sites ◽  
Aromatic Aminotransferase

In this paper we describe the cloning and sequence analysis of the tyrB and aspC genes from Escherichia coli K12, which encode the aromatic aminotransferase and aspartate aminotransferase respectively. The tyrB gene was isolated from a cosmid carrying the nearby dnaB gene, identified by its ability to complement a dnaB lesion. Deletion and linker insertion analysis located the tyrB gene to a 1.7-kilobase NruI-HindIII-digest fragment. Sequence analysis revealed a gene encoding a 43 000 Da polypeptide. The gene starts with a GTG codon and is closely followed by a structure resembling a rho independent terminator. The aspC gene was cloned by screening gene banks, prepared from a prototrophic E. coli K12 strain, for plasmids able to complement the aspC tyrB lesions in the aminotransferase-deficient strain HW225. Sub-cloning and deletion analysis located the aspC gene on a 1.8-kilobase HincII-StuI-digest fragment. Sequence analysis revealed the presence of a gene encoding a 43 000 Da protein, the sequence of which is identical with that previously obtained for the aspartate aminotransferase from E. coli B. Considerable overproduction of the two enzymes was demonstrated. We compared the deduced protein sequences with those of the pig mitochondrial and cytoplasmic aspartate aminotransferases. From the extensive homology observed we are able to propose that the two E. coli enzymes possess subunit structures, subunit interactions and coenzyme-binding and substrate-binding sites that are very similar both to each other and to those of the mammalian enzymes and therefore must also have very similar catalytic mechanisms. Comparison of the aspC and tyrB gene sequences reveals that they appear to have diverged as much as is possible within the constraints of functionality and codon usage.

scholarly journals Oligoribonuclease Is Encoded by a Highly Conserved Gene in the 3′-5′ Exonuclease Superfamily

1998 ◽  
Vol 180 (10) ◽  
pp. 2779-2781 ◽  
Author(s):  
Xiaolan Zhang ◽  
Liuqin Zhu ◽  
Murray P. Deutscher
Keyword(s):  
Kanamycin Resistance ◽  
Open Reading Frame ◽  
Sequence Information ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli ◽  
Kanamycin Resistance Cassette ◽  
Wide Range ◽  
Conserved Gene ◽  
Cloned Gene

ABSTRACT Oligoribonuclease, a 3′-to-5′ exoribonuclease specific for small oligoribonucleotides, was purified to homogeneity from extracts ofEscherichia coli. The purified protein is an α2 dimer of 40 kDa. NH2-terminal sequence analysis of the protein identified the gene encoding oligoribonuclease as yjeR(o204a), a previously reported open reading frame located at 94 min on the E. coli chromosome. However, as a consequence of the sequence information, the translation start site of this open reading frame has been revised. Cloning of yjeRled to overexpression of oligoribonuclease activity, and interruption of the cloned gene with a kanamycin resistance cassette eliminated the overexpression. On the basis of these data, we propose thatyjeR be renamed orn. Orthologs of oligoribonuclease are present in a wide range of organisms, extending up to humans.

scholarly journals Characterization of the Gene Encoding the Major Secreted Lysophospholipase A of Legionella pneumophila and Its Role in Detoxification of Lysophosphatidylcholine

2002 ◽  
Vol 70 (11) ◽  
pp. 6094-6106 ◽  
Author(s):  
Antje Flieger ◽  
Birgid Neumeister ◽  
Nicholas P. Cianciotto
Keyword(s):  
Fatty Acids ◽  
Legionella Pneumophila ◽  
Lipolytic Activity ◽  
Cholesterol Acyltransferase ◽  
Phospholipase A ◽  
Wild Type ◽  
Gene Encoding ◽  
Acyltransferase Activity ◽  
Lipolytic Enzymes ◽  
Cloned Gene

ABSTRACT We previously showed that Legionella pneumophila secretes, via its type II secretion system, phospholipase A activities that are distinguished by their specificity for certain phospholipids. In this study, we identified and characterized plaA, a gene encoding a phospholipase A that cleaves fatty acids from lysophospholipids. The plaA gene encoded a 309-amino-acid protein (PlaA) which had homology to a group of lipolytic enzymes containing the catalytic signature GDSL. In Escherichia coli, the cloned gene conferred trypsin-resistant hydrolysis of lysophosphatidylcholine and lysophosphatidylglycerol. An L. pneumophila plaA mutant was generated by allelic exchange. Although the mutant grew normally in standard buffered yeast extract broth, its culture supernatants lost greater than 80% of their ability to release fatty acids from lysophosphatidylcholine and lysophosphatidylglycerol, implying that PlaA is the major secreted lysophospholipase A of L. pneumophila. The mutant's reduced lipolytic activity was confirmed by growth on egg yolk agar and thin layer chromatography and was complemented by reintroduction of an intact copy of plaA. Overexpression of plaA completely protected L. pneumophila from the toxic effects of lysophosphatidylcholine, suggesting a role for PlaA in bacterial detoxification of lysophospholipids. The plaA mutant grew like the wild type in U937 cell macrophages and Hartmannella vermiformis amoebae, indicating that PlaA is not essential for intracellular infection of L. pneumophila. In the course of characterizing plaA, we discovered that wild-type legionellae secrete a phospholipid cholesterol acyltransferase activity, highlighting the spectrum of lipolytic enzymes produced by L. pneumophila.

An essential yeast gene encoding a TTAGGG repeat-binding protein

1993 ◽  
Vol 13 (2) ◽  
pp. 1306-1314
Author(s):  
C Brigati ◽  
S Kurtz ◽  
D Balderes ◽  
G Vidali ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Binding Activity ◽  
Insertion Mutation ◽  
Yeast Gene ◽  
Gene Encoding ◽  
Cloned Gene ◽  
Diploid Cells ◽  
Ttaggg Repeat

A yeast gene encoding a DNA-binding protein that recognizes the telomeric repeat sequence TTAGGG found in multicellular eukaryotes was identified by screening a lambda gt11 expression library with a radiolabeled TTAGGG multimer. This gene, which we refer to as TBF1 (TTAGGG repeat-binding factor 1), encodes a polypeptide with a predicted molecular mass of 63 kDa. The TBF1 protein, produced in vitro by transcription and translation of the cloned gene, binds to (TTAGGG)n probes and to a yeast telomeric junction sequence that contains two copies of the sequence TTAGGG separated by 5 bp. TBF1 appears to be identical to a previously described yeast TTAGGG-repeat binding activity called TBF alpha. TBF1 produced in vitro yields protein-DNA complexes with (TTAGGG)n probes that have mobilities on native polyacrylamide gels identical to those produced by partially purified TBF alpha from yeast cells. Furthermore, when extracts are prepared from a strain containing a TBF1 gene with an antigen tag, we find that the antigen copurifies with the predominant (TTAGGG)n-binding activity in the extracts. The DNA sequence of TBF1 was determined. The predicted protein sequence suggests that TBF1 may contain a nucleotide-binding domain, but no significant similarities to any other known proteins were identified, nor was an obvious DNA-binding motif apparent. Diploid cells heterozygous for a tbf1::URA3 insertion mutation are viable but upon sporulation give rise to tetrads with only two viable spores, both of which are Ura-, indicating that the TBF1 gene is essential for growth. Possible functions of TBF1 (TFB alpha) are discussed in light of these new results.

Glycosylated flavones as selective inhibitors of topoisomerase IV.

1997 ◽  
Vol 41 (5) ◽  
pp. 992-998 ◽  
Author(s):  
F X Bernard ◽  
S Sablé ◽  
B Cameron ◽  
J Provost ◽  
J F Desnottes ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Topoisomerase Ii ◽  
Coli Strain ◽  
Selective Inhibitors ◽  
Topoisomerase Iv ◽  
Gene Encoding ◽  
Cottonseed Flour ◽  
Dna Topoisomerase ◽  
E Coli ◽  
Pbr322 Dna

Three flavonoids which promoted Escherichia coli topoisomerase IV-dependent DNA cleavage were isolated from cottonseed flour and identified as quercetin 3-O-beta-D-glucose-[1,6]-O-alpha-L-rhamnose (rutin), quercetin 3-O-beta-D-galactose-[1,6]-O-alpha-L-rhamnose, and quercetin 3-O-beta-D-glucose (isoquercitrin). The most active one (rutin) also inhibited topoisomerase IV-dependent decatenation activity (50% inhibitory concentration, 64 microg/ml) and induced the SOS response of a permeable E. coli strain. Derivatives of quercetin glycosylated at position C-3 were shown to induce two site-specific DNA cleavages of pBR322 DNA, which were mapped by DNA sequence analysis to the gene encoding resistance to tetracycline. Cleavage at these sites was hardly detectable in cleavage reactions with quercetin or fluoroquinolones. None of the three flavonoids isolated from cottonseeds had any stimulatory activity on E. coli DNA gyrase-dependent or calf thymus topoisomerase II-dependent DNA cleavage, and they were therefore specific to topoisomerase IV. These results show that selective inhibitors of topoisomerase IV can be derived from the flavone structure. This is the first report on a DNA topoisomerase inhibitor specific for topoisomerase IV.

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