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PubMed2006

NMR observations of 13C-enriched coenzyme B12 bound to the ribonucleotide reductase from Lactobacillus leichmannii.

The 13C NMR resonance and one-bond 1H-13C coupling constants of coenzyme B12 enriched in 13C in the cobalt-bound carbon have been observed in the complex of the coenzyme with the B12-dependent ribonucleotide reductase from Lactobacillus leichmannii. Neither the 13C NMR chemical shift nor the 1H-13C coupling constants are significantly altered by binding of the coenzyme to the enzyme. The results suggest that ground-state Co-C bond distortion is not utilized by this enzyme to activate coenzyme B12 for C-Co bond homolysis.

Carbon Isotopes↗
PubMed1990

Cryo-preservation of Lactobacillus leichmannii for vitamin B12 microbiological assay.

An optimised method for preparing cryo-preserved cultures of Lactobacillus leichmannii for vitamin B12 microbiological assay is described. Cultures are mixed with an equal volume of 800 ml/l sterile glycerol before freezing. The percentage recovery of viable cells after thawing is highest when cultures are enclosed in polystyrene insulation and cooled to -70 degrees C. Cells cryo-preserved in the stationary phase of growth have a higher percentage recovery after thawing, but the same numbers of log phase cells give a larger growth response to cyanocobalamin under assay conditions. Cryo-preserved cultures are stable for many months, giving consistency of standard curve shape. Results obtained for control sera are also more consistent over time, with a range of CV values from 2.58% to 4.82%, as compared to a range of 4.63% to 6.11% for control sera in assays using conventionally prepared fresh cultures. The use of large inocula also allows for assay completion after overnight incubation.

Biological Assay↗
PubMed1993

Purification, relative molecular mass and subunits of thymidylate synthase from Lactobacillus leichmannii.

Thymidylate synthase (5, 10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) from crude cell extracts of Lactobacillus leichmannii has been purified 190-fold to homogeneity by chromatography on hydroxylapatite, DEAE-cellulose and Sephadex G-100 columns. It has UV absorption maxima at 280 nm. The crude extracts, however, have RNA associated with the native enzyme. 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]. Optimal conditions for dTMP synthase activity are: 275 microM (dl)-L-H4PteGlu, 13 mM HCHO, 13 mM MgCl2, 100 microM dUMP and 75 mM 2-mercaptoethanol at pH 7.4 using Tris-HCl buffer. The enzyme has M(r) of 74 kDa, Stokes radius of 1.24 nm and a sedimentation coefficient value of 0.45 S. The enzyme is a dimer composed of 2 identical subunits each with M(r) of 37 kDa.

Lactobacillus↗
PubMed1990

Use of beta-lactamase to hydrolyse interfering antibiotics in vitamin B12 microbiological assay using Lactobacillus leichmannii.

The presence of certain antibiotics in patients' sera can invalidate microbiological assays for vitamin B12 using Lactobacillus leichmannii by inhibiting organism growth. Analysis of 3291 consecutive serum vitamin B12 assays in a large general hospital service showed inhibition of growth in 303 samples (9.2%). In nearly all cases the interfering substance was a beta-lactam antibiotic. The value of using a beta-lactamase preparation to eliminate antibiotic effects was studied using antibiotic-containing patient sera and also normal serum to which antibiotics were added. In-vitro additions of penicillins, cephalosporins, and other antibiotics to normal serum resulted in varying levels of organism inhibition, but excellent recovery of a control value after treatment with beta-lactamase (greater than 98%). Patient sera showing inhibition were reassayed in duplicate following treatment with beta-lactamase (n = 200), the overall effectiveness of the treatment being verified by the excellent recovery (greater than 98%) of a cyanocobalamin 'spike' added to a control. In only 0.2% of cases (6 of 3291 samples) was this treatment ineffective and it has thus proved an efficient method of eliminating the problem of antibiotic interference in the vitamin B12 microbiological assay.

Anti-Bacterial Agents↗
PubMed1998

Gemcitabine 5'-triphosphate is a stoichiometric mechanism-based inhibitor of Lactobacillus leichmannii ribonucleoside triphosphate reductase: evidence for thiyl radical-mediated nucleotide radical formation.

Ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii utilizes adenosylcobalamin and catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates. One equivalent of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate, F2dCTP, rapidly inactivates RTPR. Analysis of the reaction products reveals that inactivation is accompanied by release of two fluoride ions and 0.84 equiv of 5'-deoxyadenosine and attachment of 1 equiv of corrin covalently to an active-site cysteine residue of RTPR. No cytosine release was detected. Proteolysis of corrin-labeled RTPR with endoproteinase Glu-C and peptide mapping at pH 5.8 revealed that C419 was predominantly modified. The kinetics of the inactivation have been examined by stopped-flow (SF) UV-vis spectroscopy and rapid freeze quench (RFQ) electron paramagnetic resonance (EPR) spectroscopy. Monitoring DeltaA525 nm shows that cob(II)alamin is formed with an apparent kobs of 50 s-1, only 2. 5-fold slower than a similar experiment carried out with cytidine 5'-triphosphate (CTP). The same reaction mixture was thus quenched at times from 22 ms to 30 s and examined by EPR spectroscopy. At early time points the EPR spectrum resembled a thiyl radical exchange coupled to cob(II)alamin. From 22 to 255 ms the total spin concentration remained unchanged at 1.4 spins/RTPR, twice that predicted by the amount of cob(II)alamin determined by SF. However, with time the signal attributed to the thiyl radical-cob(II)alamin disappears and new signal(s) with broad feature(s) at g = 2.33 and a sharp feature at g = 2.00 appeared, suggesting formation of cob(II)alamin and a nucleotide-based radical with only dipolar interactions. These studies have been interpreted to support the proposal that an RTPR-based thiyl radical can give rise to a nucleotide-based radical.

Cytosine↗
PubMed1983

Interaction of 3'-[3H]2'-Chloro-2'-deoxyuridine 5'-triphosphate with ribonucleotide reductase from Lactobacillus leichmannii.

Incubation of [3'-3H]2'-chloro-2'-deoxyuridine 5'-triphosphate (CldUTP) with adenosylcobalamin (AdoCbl), reductant, and ribonucleotide reductase from Lactobacillus leichmannii results in the production of 3H2O, uracil, tripolyphosphate, 5'-deoxyadenosine, and cob(II)alamin. The rate of 3H2O release (0.19 mumol/min/mg) is almost identical with the rate of UTP reduction (0.24 mumol/min/mg). The amount of 3H2O release is dependent upon the enzyme to cofactor ratio. With a ribonucleotide reductase: AdoCbl ratio of 1:1000, approximately 500 eq of 3H2O are released. At this time the enzyme is still active, but further destruction of cofactor and turnover of CldUTP is prevented by competitive inhibition of Co(II) + 5'-deoxyadenosine with AdoCbl for binding to ribonucleotide reductase. The 5'-deoxyadenosine and AdoCbl reisolated during incubation of [3'-3H]CldUTP and ribonucleotide reductase contains no detectable radioactivity.

Cobamides↗
PubMed1994

Physico-chemical properties of thymidylate synthase from Lactobacillus leichmannii: thiol groups, amino acid composition and the terminal end-groups.

Thymidylate synthase (5,10-methylenetetrahydrofolate: deoxyuridylate C-methyltransferase, EC 2.1.1.45) from Lactobacillus leichmannii was completely inactivated after 5 min of heat treatment at 55 degrees C. A remarkable synergistic effect with no loss in activity was noted when 10(-3) M dUMP was added to the enzyme before subjecting to heat treatment. The enzyme got activated in the presence of 2-mercaptoethanol (75 mM) and inhibited by pCMB (I50 = 5 microM). It had 2 free sulfhydryl groups and a single disulfide bond. The two identical subunits of the 74 kDa dimer were possibly bonded by a single disulfide linkage. It had a total of 652 amino acids with methionine as the amino-terminal and alanine as the carboxy-terminal amino acid residues. The carboxy-terminal end-group alanine was preceded by valine, lysine and proline sequentially in that order.

Amino Acids↗
PubMed2000

Purification and characterization of dihydrofolate reductase from Lactobacillus leichmannii.

Dihydrofolate reductase (DHFR) (5,6,7,8-THF: NADDP+ oxidoreductase, EC 1.5.1.3) was purified 205-fold to apparent homogeneity from the crude extracts of Lactobacillus leichmannii. It has UV absorption maxima at 280 nm, M(r) of 20,000, Stokes radius of 0.34 nm and a S20.w value of 0.12 S. The preparation showed the presence of 168 amino acid residues with threonine and lysine as the NH2- and COOH- terminal end-groups respectively and a single reactive sulfhydryl group. pCMB inhibited the enzyme activity (IC50 = 2 microM). The enzyme has a pH optimum of 7.4 and is thermally inactivated at > 35 degrees C. It is activated by 0.1 M KCl and KI and 2 M urea. 3-4 M urea completely inactivated the enzyme. Enzyme has Km values of 3.5 microM and 6.2 microM for NADPH and DHF respectively, and a Ki value of 7 nM for MTX, the inhibition being competitive.

Amino Acids↗
PubMed1999

Adenosylcobalamin-dependent ribonucleoside triphosphate reductase from Lactobacillus leichmannii. Rapid, improved purification involving dGTP-based affinity chromatography plus biophysical characterization studies demonstrating enhanced, "crystallographic level" purity.

Ribonucleoside triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacillus leichmannii is a 5'-deoxyadenosylcobalamin-dependent (AdoCbl; Coenzyme B12) enzyme. RTPR is also a prototypical adenosylcobalamin-dependent ribonucleotide reductase, one that, as its name indicates, converts ribonucleoside triphosphates (NTP) to deoxyribonucleoside triphosphates (dNTP). Upon substrate binding to RTPR, AdoCbl's cobalt-carbon bond is cleaved to generate cob(II)alamin, 5'-deoxyadenosine, and the cysteine (C408) derived thiyl radical. Five key cysteines (Cys 119, 408, 419, 731, and 736), from among the ten total cysteines, are involved in RTPR's catalytic mechanism. A critical examination of the RTPR isolation and purification literature suggested that the purification protocol currently used results in RTPR which contains 2040% microheterogeneity, along with minor contamination by other proteins. In addition, no report of crystalline RTPR has ever appeared. The literature indicates that irreversible cysteine oxidation (e.g., to -SO2H or -SO3H) is one highly plausible reason for the microheterogeneity of RTPR. The literature also indicates that improvement in the level of enzyme purity is the most effective next step in coaxing enzymes to crystallize that have previously failed to do so. A shortened, improved purification of RTPR has been developed, one involving a shorter purification time, a lower pH, a higher concentration of the more effective reductant DTT (all designed to help protect the cysteines from oxidation), and a final step utilizing our recently reported, improved dGTP-based affinity chromatography resin. The resultant RTPR is approximately 20-30% higher in both specific activity and in its ability to undergo single turnovers, and is homogeneous by mass spectrometry and dynamic light scattering. Additionally, the revised purification procedure eliminates > 30 proteins present in 2-3% amounts along with damaged RTPR that does not bind properly (i.e. tightly) to the dGTP-affinity resin. Finally, dGTP-based affinity chromatography purified RTPR has yielded the first reported, albeit small, single crystals of RTPR.

Allosteric Regulation↗
PubMed1988

Inactivation of the ribonucleoside triphosphate reductase from Lactobacillus leichmannii by 2'-chloro-2'-deoxyuridine 5'-triphosphate: a 3'-2' hydrogen transfer during the formation of 3'-keto-2'-deoxyuridine 5'-triphosphate.

The ribonucleoside triphosphate reductase of Lactobacillus leichmannii converts the substrate analogue 2'-chloro-2'-deoxyuridine 5'-triphosphate (ClUTP) into a mixture of 2'-deoxyuridine triphosphate (dUTP) and the unstable product 3'-keto-2'-deoxyuridine triphosphate (3'-keto-dUTP). This ketone can be trapped by reduction with NaBH4, producing a 4:1 mixture of xylo-dUTP and dUTP. When [3'-3H]ClUTP is treated with enzyme in the presence of NaBH4, the isomeric deoxyuridines isolated after alkaline phosphatase treatment retained 15% of the 3H in ClUTP. Degradation of these isomeric nucleosides has established the location of the 3H in 3'-keto-dUTP as predominantly 2'(S). The xylo-dU had 98.6% of its label at the 2'(S) position and 1.5% at 2'(R). The isolated dU had 89.6% of its label at 2'(S) and 1.4% at 2'(R), with the remaining 9% label inferred to be at the 3'-carbon, this resulting from the direct enzymic production of dUTP. These results are consistent with enzymic production of a 1:1000 mixture of dUTP and 3'-keto-dUTP, where the 3'-hydrogen of ClUTP is retained at 3' during production of dUTP and is transferred to 2'(S) during production of 3'-keto-dUTP. The implications of these results and the unique role of the cofactor adenosylcobalamin (Ashley et al., 1986) are discussed in terms of reductase being a model for the B12-dependent rearrangement reactions.

Deoxyuracil Nucleotides↗
PubMed1988

Inactivation of the Lactobacillus leichmannii ribonucleoside triphosphate reductase by 2'-chloro-2'-deoxyuridine 5'-triphosphate: stoichiometry of inactivation, site of inactivation, and mechanism of the protein chromophore formation.

The ribonucleoside triphosphate reductase (RTPR) of Lactobacillus leichmannii is inactivated by the substrate analogue 2'-chloro-2'-deoxyuridine 5'-triphosphate (ClUTP). Inactivation is due to alkylation by 2-methylene-3(2H)-furanone, a decomposition product of the enzymic product 3'-keto-2'-deoxyuridine triphosphate. The former has been unambiguously identified as 2-[(ethylthio)methyl]-3(2H)-furanone, an ethanethiol trapped adduct, which is identical by 1H NMR spectroscopy with material synthesized chemically. Subsequent to rapid inactivation, a slow process occurs that results in formation of a new protein-associated chromophore absorbing maximally near 320 nm. The terminal stages of the inactivation have now been investigated in detail. The alkylation and inactivation stoichiometries were studied as a function of the ratio of ClUTP to enzyme. At high enzyme concentrations (0.1 mM), 1 equiv of [5'-3H]ClUTP resulted in 0.9 equiv of 3H bound to protein and 83% inactivation. The amount of labeling of RTPR increased with increasing ClUTP concentration up to the maximum of approximately 4 labels/RTPR, yet the degree of inactivation did not increase proportionally. This suggests that (1) RTPR may be inactivated by alkylation of a single site and (2) decomposition of 3'-keto-dUTP is not necessarily enzyme catalyzed. The formation of the new protein chromophore was also monitored during inactivation and found to reach its full extent upon the first alkylation. Thus, out of four alkylation sites, only one appears capable of undergoing the subsequent reaction to form the new chromophore. While chromophore formation was prevented by NaBH4 treatment, the chromophore itself is resistant to reduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1981

On the mechanism of ribonucleoside triphosphate reductase from Lactobacillus leichmannii. Evidence for 3' C--H bond cleavage.

The 3' carbon-hydrogen bond of [3'-3H]uridine 5'-triphosphate is is cleaved during its conversion to 2'-deoxyuridine 5'-triphosphate catalyzed by Lactobacillus leichmannii ribonucleoside triphosphate reductase. A selection against 3H of approximately 1.8 is observed on this reduction. During the course of this reaction, no 3H is released to the solvent, and no 3H is recovered in reisolated coenzyme B12. Incubation of [3'-2H]uridine 5'-triphosphate with enzyme resulted in production of 2'-deoxy[3'-2H]uridine 5'-triphosphate.

Carbon Radioisotopes↗
PubMed2001

Inactivation of Escherichia coli O1 57:H7, Listeria monocytogenes, and Lactobacillus leichmannii by combinations of ozone and pulsed electric field.

Pulsed electric field (PEF) and ozone technologies are nonthermal processing methods with potential applications in the food industry. This research was performed to explore the potential synergy between ozone and PEF treatments against selected foodborne bacteria. Cells of Lactobacillus leichmannii ATCC 4797, Escherichia coli O157:H7 ATCC 35150, and Listeria monocytogenes Scott A were suspended in 0.1% NaCl and treated with ozone, PEF, and ozone plus PEE Cells were treated with 0.25 to 1.00 microg of ozone per ml of cell suspension, PEF at 10 to 30 kV/cm, and selected combinations of ozone and PEF. Synergy between ozone and PEF varied with the treatment level and the bacterium treated. L. leichmannii treated with PEF (20 kV/cm) after exposure to 0.75 and 1.00 microg/ml of ozone was inactivated by 7.1 and 7.2 log10 CFU/ml, respectively; however, ozone at 0.75 and 1.00 microg/ml and PEF at 20 kV/cm inactivated 2.2, 3.6, and 1.3 log10 CFU/ml, respectively. Similarly, ozone at 0.5 and 0.75 microg/ml inactivated 0.5 and 1.8 log10 CFU/ml of E. coli, PEF at 15 kV/cm inactivated 1.8 log10 CFU/ml, and ozone at 0.5 and 0.75 microg/ml followed by PEF (15 kV/cm) inactivated 2.9 and 3.6 log10 CFU/ml, respectively. Populations of L. monocytogenes decreased 0.1, 0.5, 3.0, 3.9, and 0.8 log10 CFU/ml when treated with 0.25, 0.5, 0.75, and 1.0 microg/ml of ozone and PEF (15 kV/cm), respectively; however, when the bacterium was treated with 15 kV/cm, after exposure to 0.25, 0.5, and 0.75 microg/ml of ozone, 1.7, 2.0, and 3.9 log10 CFU/ml were killed, respectively. In conclusion, exposure of L. leichmannii, E. coli, and L. monocytogenes to ozone followed by the PEF treatment showed a synergistic bactericidal effect. This synergy was most apparent with mild doses of ozone against L. leichmannii.

Colony Count, Microbial↗
PubMed1979

Mechanism of Lactobacillus leichmannii ribonucleotide reductase studied with Coalpha-[alpha-(Aden-9-yl)]-Cobeta-adenosylcobamide (Pseudocoenzyme B12) as coenzyme.

Coalpha-[alpha-(Aden-9-yl)]-Cobeta-adenosylcobamide (pseudocoenzyme B12) purified from Clostridium tetanomorphum has been reacted with ribonucleotide reductase purified from Lactobacillus leichmannii under various conditions, and the properties of the products obtained have been compared by electron paramagnetic resonance (EPR) with those previously reported for products formed from the normal coenzyme (adenosylcobalamin). The rapidly formed intermediate and the slowly formed "doublet" species from the pseudocoenzyme have EPR spectra identical with those formed from the normal coenzyme. This and other considerations make it less likely that the unusual magnetic properties of the rapidly formed intermediate are due to strongly distorted octahedral symmetry about Co(II) as previously postulated. Instead it is probable that the EPR spectrum is due to interaction of the radical pair by both exchange coupling and magnetic dipole--dipole coupling. Although Coalpha-[alpha-(aden-9-YL)]cob(II)amide in solution does not show superhyperfine splitting in the EPR spectrum because of its base-off configuration, the cob(II)amide formed by degradation of the pseudocoenzyme within the catalytic site of the enzyme did show triplets due to a nitrogen axially coordinated to cobalt. This suggests that binding of the cob(II)amide to the reductase catalytic site causes a shift to the base-on form.

Electron Spin Resonance Spectroscopy↗
PubMed1994

Pteroyl- and tetrahydropteroylpolyglutamate effects on the catalytic activity of thymidylate synthase from Lactobacillus leichmannii: a novel method for determining gamma-glutamyl chain lengths of the folylpolyglutamates.

Tetrahydropteroylpolyglutamates with longer gamma-glutamyl chain lengths have been found to act as better cofactors than the corresponding monoglutamates for the activity of thymidylate synthase, (5, 10-CH2H4PteGlu: dUMP C-methyltransferase, EC 2.1.1.45) purified from Lactobacillus leichmannii. Contrarily, the pteroylpolyglutamates (unreduced forms) with longer gamma-glutamyl chain lengths act as powerful inhibitors of the same enzyme, the I50 being 2 microM for the tetraglutamate, and inhibition is competitive. The Km and Ki values for the synthetic folylpolyglutamates are identical to those obtained for the natural folylpolyglutamyl forms isolated from Torula yeast (Candida utilis) by the author earlier. A rapid novel method is suggested that could be conveniently used to determine the gamma-glutamyl chain lengths of the folylpolyglutamates employing the direct or indirect linear proportionality relationship observed between the number of gamma-glutamyl residues linked and the Ki and Km values of the enzyme considering the state of oxidation/reduction of the pteridine moiety and the 1-C substituents attached.

Catalysis↗
PubMed1975

An affinity adsorbent containing deoxyguanosine 5'-triphosphate linked to sepharose and its use for large scale preparation of ribonucleotide reductase of Lactobacillus leichmannii.

P3-(6-(N-Trifluoracetyl)aminohex-1-yl) deoxyguanosine triphosphate has been prepared by the reaction of N-trifluoroacetyl-6-aminohexanol 1-pyrophosphate with the imidazolide of dGMP and has been characterized. This compound and the corresponding free amine, obtained by removal of the protective trigluoroacetyl group, are activators of ribonucleotide reductase of Lactobacillus leichmannii. An affinity adsorbent for the reductase, prepared by reaction of the amine derivative with CNBr-activated Sepharose, contains dGTP covalently attached through the gamma-phosphate via a six-carbon chain to the matrix. The method of synthesis of the dGTP derivative is generally applicable to the synthesis of P3-(omega-aminoalk-1-yl)nucleoside triphosphate esters for the preparation of analogous affinity adsorbents. Ribonucleotide reductase can be rapidly purified to homogeneity, on a large scale, by use of dGTP-Sepharose and conditions for optimum recovery of the enzyme have been determined. The affinity of ribonucleotide reductase and other proteins for dGTP-Sepharose is increased by either raising the ionic strength or lowering the temperature of the eluent. Elution of the enzyme from the adsorbent can be achieved between pH 5.8 and 7.3, whereas at pH 5.3 the reductase is bound extremely tightly and cannot be recovered. Ribonucleotide reductase can be eluted from the adsorbent with dGTP or urea. Elution with urea is carried out at pH 6.3, where the enzyme is stable and maximum recovery is obtained. Affinity chromatography consistently produces ribonucleotide reductase of high specific activity (170-180 units/mg). In the presence of 0.1 to 1.2 M urea or hydroxyurea, the enzyme is inhibited, but allosteric activation is unchanged. No alteration in the structure or function of the reductase was detected when the enzyme was exposed to 2.0 M urea during elution from the affinity adsorbent, but exposure for longer periods causes some inactivation.

Amino Acid Sequence↗