PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “LACTOBACILLUS LEICHMANNII”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1993

Ternary complex formation with 5-fluoro-2'-deoxyuridylate and the kinetic properties of thymidylate synthase from Lactobacillus leichmannii.

Lactobacillus leichmannii thymidylate synthase (5,10-CH2-H4PteGlu:dUMP C-methyltransferase, EC 2.1.1.45) forms a tight and stable covalently bonded ternary complex with the inhibitor 5-FdUMP in the presence of the cofactor 5,10-CH2-H4-PteGlu. 'Filter assay' employing the radioactive nucleotide ligand showed that 2 moles of FdUMP are bound per mole enzyme during the ternary complex formation with the L. leichmannii dTMP synthase. This is in line with our earlier observation on the Streptococcus faecium thymidylate synthase [Narasimha Rao, K & Kisliuk R L (1983), Proc Natl Acad Sci USA, 80, 916-920]. The enzyme has Km values of 6.3 x 10(-6) M, 8.2 x 10(-5) M and 1.0 x 10(-4) M for dUMP, (dl)-L-H4PteGlu and Mg2+ respectively; Vmax for dUMP, (dl)-L-H4PteGlu and Mg2+ are; 0.55, 0.5 and 1.1 respectively. It has K(i) values of 6.7 x 10(-6) M, 2.2 x 10(-6) M, 5.0 x 10(-5) M and 2.0 x 10(-4) M for FdUMP, dTMP, MTX and Ca2+ respectively. The type of enzyme inhibition with FdUMP, dTMP, MTX and Ca2+ was competitive. dTMP Studies clearly show the 'end product' inhibition of the enzyme.

Binding Sites↗
PubMed1973

Evidence for four deoxynucleoside kinase activities in extracts of Lactobacillus leichmannii.

Extracts of Lactobacillus leichmannii (ATCC 7830) catalyze the phosphorylation of the four principal deoxynucleosides. Thymidine, deoxyguanosine, and deoxycytidine kinase activities were found to be optimal with deoxyadenosine triphosphate as the phosphoryl donor, whereas deoxycytidine triphosphate was the optimal donor for deoxyadenosine kinase activity. L. leichmannii catalyzes the conversion of deoxycytidine to deoxyuridylic acid, probably by a pathway involving deoxycytidylate deaminase.

Cell-Free System↗
PubMed1996

Molecular cloning and characterization of the pyrB gene of Lactobacillus leichmannii encoding aspartate transcarbamylase.

The Lactobacillus leichmannii pyrB gene, encoding pyrimidine biosynthetic enzyme aspartate transcarbamylase (ATCase), was cloned from a partial genomic library lying on a 1468 bp Sa/I/BstXI fragment. The predicted polypeptide sequence extending over 351 amino acid residues (M(r) 39 855 Da) was compared to those of various other organisms revealing clear identities towards them and important conservative stretches, implying that these proteins are closely related. Transcriptional initiation was mapped by primer extension and occurred 54 bp upstream of the pyrB open reading frame (ORF). Northern blot analysis indicates that the pyrB gene is transcribed as a single mRNA and not together with the following overlapping pyrC gene as a bicistronic mRNA. At high copy number the pyrB gene of L leichmannii seems to be lethal for its E coli host; inserted in a low copy vector it complements the uracil auxotrophy of an E coli pyrB mutant which shows distinct ATCase activity in the cell extract. With an excess of uracil in the growth medium the gene is apparently repressed and no ATCase activity can be measured.

Amino Acid Sequence↗
PubMed1998

The function of adenosylcobalamin in the mechanism of ribonucleoside triphosphate reductase from Lactobacillus leichmannii.

Ribonucleoside triphosphate reductase from Lactobacillus leichmannii catalyzes the reduction of nucleotides to deoxynucleotides and uses adenosylcobalamin as a cofactor. A transient protein-based thiyl radical is essential for catalysis. Studies directed toward the elucidation of the function of adenosylcobalamin during catalysis have shown that formation of the thiyl radical and 5'-deoxyadenosine occurs in a concerted fashion with C-Co bond homolysis, that the homolysis is entropically and not enthalpically driven, that the dimethylbenzimidazole moiety of adenosylcobalamin is the axial ligand during catalysis, and that the C-Co bond is reformed after every turnover.

Allosteric Regulation↗
PubMed1995

Cloning, sequencing, and characterizing the Lactobacillus leichmannii pyrC gene encoding dihydroorotase.

The gene encoding dihydroorotase (DHOase) of Lactobacillus leichmannii, the third enzyme of the pyrimidine biosynthetic pathway (Genbank (EMBL) accession no X78999), was cloned by phenotypic complementation of an E coli pyrC deficient mutant after transformation with Lactobacillus leichmannii genomic library DNA. The open reading frame of the L leichmannii pyrC gene spans 1281 bp and codes for a 427 amino cid polypeptide with a calculated M(r) of 46,316 Da. Primer extension showed that the initiation site for transcription is 37 bp upstream of the putative start codon ATG and Northern blot analysis confirmed its independent transcription from the adjacent pyrB gene. Comparison of the deduced amino acid sequence of L leichmannii DHOase with sequences established for other organisms yielded 46.6% identity with the corresponding Bacillus subtilis enzyme. Highly conserved protein domains suggest importance for the enzyme's function.

Amino Acid Sequence↗
PubMed1968

Pyrimidine biosynthesis in Lactobacillus leichmannii.

Tracer studies of pyrimidine biosynthesis in Lactobacillus leichmannii (ATCC 7830) indicated that, while aspartate is utilized in the usual manner, the guanido carbon of arginine, rather than carbon dioxide, is utilized as a pyrimidine precursor. The guanido carbon of arginine also contributes, to some extent, to the carbon dioxide pool utilized for purine biosynthesis. The enzyme of the first reaction leading from arginine to pyrimidines, arginine deiminase, was investigated in crude bacterial extracts. It was inhibited by thymidylic acid and purine ribonucleotides, and to a lesser extent by purine deoxynucleotides and deoxycytidylic acid. Under the assay conditions employed, a number of nucleotides had no effect on the enzyme activity of the aspartate transcarbamylase of L. leichmannii. Growth of the cells in media containing uracil, compared to growth in media without uracil, resulted in a four- to fivefold decrease in the concentrations of aspartate transcar-bamylase and dihydroorotase and a twofold increase in the concentration of arginine deiminase, as estimated from specific enzyme activity in crude extracts of the cells. A small increase in specific enzyme activity of ornithine transcarbamylase and carbamate kinase was also observed in extracts obtained from cells grown on uracil. No appreciable change in concentration of any of the five enzymes studied was detected when the cells were grown in media containing thymidine or guanylic acid. A hypothetical scheme which suggests a relationship between the control of purine and pyrimidine biosynthesis in this bacterium and which is consistent with the experimental results obtained is presented.

Arginine↗
PubMed1996

Rapid purification and characterization of two distinct N-deoxyribosyltransferases of Lactobacillus leichmannii.

Two distinct N-deoxyribosyltransferases of Lactobacillus leichmannii, designated as DRTase I and DRTase II, were separated and purified almost to homogeneity by one-step affinity chromatography. DRTase I is distinguished by specifically catalyzing the direct transfer of 2-deoxyribosyl residues from purine deoxyribonucleosides to free purine bases, whereas DRTase II has a rather broad substrate specificity and is able to transfer the deoxyribosyl moiety between pyrimidines and between purines and pyrimidines. Furthermore, in addition to the different substrate spectrum, we clearly differentiated the two enzymes by comparing their varying temperature/activity and pH/activity profiles, their kinetic constants, their behaviour in Western blot analysis, and their N-terminal amino acid sequences. Denaturing and non-denaturing DISK-PAGE revealed strong evidence that both intact enzymes consist of hexamers with subunit molecular weights of approximately 20,000 for DRTase I and 18,000 for DRTase II.

Adenine↗
PubMed1965

SURVIVAL OF LACTOBACILLUS LEICHMANNII IN RELATION TO VITAMIN B12 ASSAYS.

Since washed cells of Lactobacillus leichmannii ATCC 7830 frequently showed erratic results in vitamin B(12) assays, a depletion technique was used to stabilize the inoculum. The method consisted of incubating the twice-washed cells at 37 C for 45 min in distilled water. Survival and growth studies indicated that the stabilizing procedure (i) did not affect cell numbers, (ii) was optimal for depleting possible carry-over of vitamin B(12) or other nutrilites and reserves, and (iii) brought about a phasing of the cells of the inoculum which underwent logarithmic growth immediately on inoculation. This minimal period (45 min) was the same when the cells were incubated in dextrose-water. Survival of the cells in physiological saline was greater than in distilled water, but a longer time was required for stabilizing the inoculum in saline, which precluded its use in routine work. The prepared inoculum showed an improvement over the washed inoculum in that readings between duplicate and triplicate tubes agreed closely and the standard curves were reproducible whether acidimetric or turbidimetric methods were followed. No erratic results were noted in more than 1 year of testing.

Biological Assay↗
PubMed1964

LIQUID NITROGEN FREEZING IN MICROBIOLOGICAL ASSAY SYSTEMS. I. PRESERVATION OF LACTOBACILLUS LEICHMANNII FOR DIRECT USE IN THE VITAMIN B12 ASSAY.

Suspensions of Lactobacillus leichmannii were stored in liquid nitrogen and were used as direct inocula in vitamin B(12) assays. Complete recovery of viable cells was obtained when the suspensions in basal B(12) medium were rapidly frozen by direct immersion into liquid nitrogen and rapidly thawed by agitating the suspensions in a water bath at 40 C. Greater than 90% destruction occurred when the suspensions were in saline. However, both suspensions were usable in the B(12) assay system. Assay results on a number of test materials indicated good correlation between freshly prepared suspensions and frozen suspensions in basal medium stored 3 months. Suspensions in saline stored for 1 year in liquid nitrogen showed no detectable difference from the first day after freezing. Suspensions frozen slowly at the rate of 1 degree per min from 4 to -40 C and subsequently immersed in liquid nitrogen had a longer lag period of growth and were not usable in the 18-hr assay incubation system. A major advantage of a stored inoculum for direct use in a microbiological assay is the reduced day-to-day variation in the inoculum.

Biological Assay↗
PubMed1993

Cloning, sequencing, and expression of the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii.

Ribonucleoside-triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacillus leichmannii, a monomeric adenosylcobalamin-requiring enzyme, catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates. The gene for this enzyme has been cloned and sequenced. In contrast to expectations based on mechanistic considerations, there is no statistically significant sequence homology with the Escherichia coli reductase that requires a dinuclear-iron center and tyrosyl radical cofactor. The RTPR has been overexpressed and purified to homogeneity, yielding 90 mg of protein from 2.5 g of bacteria. Initial characterization of the recombinant RTPR indicates that its properties are identical to those of the RTPR isolated from L. leichmannii.

Amino Acid Sequence↗
PubMed1998

Reversible inhibition by pyridoxal 5'-phosphate and irreversible inactivation by urea and guanidine hydrochloride of thymidylate synthase from Lactobacillus leichmannii.

Pyridoxal 5'-phosphate reversibly inhibited thymidylate synthase from Lactobacillus leichmannii. The inhibition was competitive with dUMP (Ki = 1 microM) and non-competitive with 5,10-CH2-THF (Ki = 0.08 microM). Treatment of native or pCMB-treated enzyme with urea (5 M) or guanidine hydrochloride (4 M) resulted in inactivation and dissociation of the homodimer (74 kDa) into monomer (37 kDa).

Dimerization↗
PubMed1975

Extraction of serum vitamin B12 for radio-isotopic and Lactobacillus leichmannii assay.

The protein precipitates discarded during the extraction process of the Lactobacillus leichmannii vitamin B12 assay have been shown to contain significant amounts of vitamin B12. This loss of vitamin B12 provide a satisfactory explanation for many of the descrepancies between the serum vitamin B12 values obtained by the L. leichmannii method and the radio-isotopic method of Raven et al (1969). It is possible to produce lower results by the method of Raven et al (1969)by incorporating into that method the L. leichmannii extraction process; it is also possible to produce higher results by the L. leichmannii method using a papain extraction process.

Lactobacillus↗
PubMed1987

2'-Deoxy-2'-halonucleotides as alternate substrates and mechanism-based inactivators of Lactobacillus leichmannii ribonucleotide reductase.

The interaction of the ribonucleoside-triphosphate reductase of Lactobacillus leichmannii with various 2'-halogenated ribo- and arabinonucleoside triphosphates has been investigated. All analogues examined acted as mechanism-based inactivators of the enzyme, producing base, triphosphate, and halide. In all cases, the inactive enzyme had developed the distinctive chromophore at 320 nm that is characteristic of enzyme inactivated by 2-methylene-3(2H)-furanone. The striking similarities between these results and those previously reported for the inactivation of this enzyme by 2'-chloro-2'-deoxyuridine triphosphate suggest a common reaction path for all 2'-halonucleotides. In the pyrimidine series, it was found that 2'-fluoro- and 2'-chloronucleotides partitioned between inactivation and formation of the normal reduction product 2'-deoxynucleotide. Normal reduction predominated with 2'-fluoronucleotides, whereas it was a minor pathway for 2'-chloro-2'-deoxyuridine triphosphate. With 2'-chloro-2'-deoxyuridine triphosphate, the relative partitioning between the two modes was pH dependent: the amount of 2'-deoxyuridine triphosphate formed increased 2.8-fold upon changing from pH 6.1 to pH 8.3. The ability of 2'-arabinohalonucleotides to inactivate ribonucleotide reductase and the variation of partitioning of the pyrimidine analogues with leaving group and reaction pH are consistent with our radical cation hypothesis and support the proposal that the difference between normal catalysis and inactivation is related to the protonation state of the reductase.

Deoxyribonucleotides↗
PubMed1999

Binding of Cob(II)alamin to the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii. Identification of dimethylbenzimidazole as the axial ligand.

The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleoside 5'-triphosphates to 2'-deoxynucleoside 5'-triphosphates and uses coenzyme B12, adenosylcobalamin (AdoCbl), as a cofactor. Use of a mechanism-based inhibitor, 2'-deoxy-2'-methylenecytidine 5'-triphosphate, and isotopically labeled RTPR and AdoCbl in conjunction with EPR spectroscopy has allowed identification of the lower axial ligand of cob(II)alamin when bound to RTPR. In common with the AdoCbl-dependent enzymes catalyzing irreversible heteroatom migrations and in contrast to the enzymes catalyzing reversible carbon skeleton rearrangements, the dimethylbenzimidazole moiety of the cofactor is not displaced by a protein histidine upon binding to RTPR.

Benzimidazoles↗
PubMed1987

Location of the redox-active thiols of ribonucleotide reductase: sequence similarity between the Escherichia coli and Lactobacillus leichmannii enzymes.

The redox-active thiols of Escherichia coli ribonucleoside diphosphate reductase and of Lactobacillus leichmannii ribonucleoside triphosphate reductase have been located by a procedure involving (1) prereduction of enzyme with dithiothreitol, (2) specific oxidation of the redox-active thiols by treatment with substrate in the absence of exogenous reductant, (3) alkylation of other thiols with iodoacetamide, and (4) reduction of the disulfides with dithiothreitol and alkylation with [1-14C]iodoacetamide. The dithiothreitol-reduced E. coli B1 subunit is able to convert 3 equiv of CDP to dCDP and is labeled with 5.4 equiv of 14C. Sequencing of tryptic peptides shows that 2.8 equiv of 14C is on cysteines-752 and -757 at the C-terminus of B1, while 1.0-1.5 equiv of 14C is on cysteines-222 and -227. It thus appears that two sets of redox-active dithiols are involved in substrate reduction. The L. leichmannii reductase is able to convert 1.1 equiv of CTP to dCTP and is labeled with 2.1 equiv of 14C. Sequencing of tryptic peptides shows that 1.4 equiv of 14C is located on the two cysteines of C-E-G-G-A-C-P-I-K. This peptide shows remarkable and unexpected similarity to the thiol-containing region of the C-terminal peptide of E. coli B1, C-E-S-G-A-C-K-I.

Alkylation↗
PubMed1994

Folylpolyglutamate synthetase in Lactobacillus leichmannii: in vitro synthesis and characterization of polyglutamylfolates.

In vitro synthesis of folylpolyglutamates by folylpolyglutamate synthetase from Lactobacillus leichmannii has been studied and optimal conditions for enzyme activity determined. It is found that while ATP (5 mM) is essential for the synthesis of folylpolyglutamates homocysteine augments the same. Replacement of vitamin B12 (2 ng/ml) with deoxyuridine (20 micrograms/ml) in growth medium does not alter the enzymatic parameters studied. DEAE-cellulose column chromatography of in vitro synthesised folylpolyglutamates indicates that folylpolyglutamate synthetase of L. leichmannii can synthesize polyglutamates up to a chain length of four glutamate residues.

Lactobacillus↗