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R Banerjee

Publications and source records attributed to R Banerjee.

At least 145 records · Page 8Linked to original sources
PubMed1996

Cloning, mapping and RNA analysis of the human methionine synthase gene.

Elevated levels of plasma homocysteine is a risk factor in both birth defects and vascular disease. Methionine synthase (MS) is a cobalamin dependent enzyme which catalyzes methylation of homocysteine to methionine. Impaired MS activity is expected to lead to increased levels of plasma homocysteine. In addition, defects in this gene may underlie the methionine-dependence observed in a number of human tumor cell lines. We describe here the isolation and characterization of the human MS cDNA. It contains an open reading frame of 3798 nucleotides encoding a protein of 1265 amino acids with a predicted molecular mass of 140 kDa. The amino acid sequence of the human MS is 55% identical with that of the Escherichia coli enzyme (METH) and 64% identical with the predicted Caenorhabditis elegans enzyme. Seven peptide sequences derived from purified porcine MS have substantial similarity to the human protein. Northern analysis indicates that the MS RNA is present in a wide variety of tissues. We have mapped the human gene to chromosomal location 1q43, a region found monosomic in individuals with deletion 1q syndrome. The isolation of the MS cDNA will now allow the direct determination of whether mutations in this gene contribute to folate-related neural tube defects, cardiovascular diseases, and birth defects.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1996

Defects in human methionine synthase in cblG patients.

Inborn errors resulting in isolated functional methionine synthase deficiency fall into two complementation groups, cblG and cblE. Using biochemical approaches we demonstrate that one cblG patient has greatly reduced levels of methionine synthase while in another, the enzyme is specifically impaired in the reductive activation cycle. The biochemical data suggested that low levels of methionine synthase activity in the first patient may result from mutations in the catalytic domains of the enzyme, reduced transcription, or generation of unstable message or protein. Using Northern analysis, we demonstrate that the molecular basis for the biochemical phenotype in this patient is associated with greatly diminished steady-state levels of methionine synthase mRNA. The biochemical data on the second patient cell line implicated mutations specific to reductive activation, a function that is housed in the C-terminal AdoMet-binding domain and the intermediate B12-binding domain, in the highly homologous bacterial enzyme. We have detected two mutations in a compound heterozygous state, one that results in conversion of a conserved proline (1173) to a leucine residue and the other a deletion of an isoleucine residue (881). The crystal structure of the C-terminal domain of the Escherichia coli MS predicts that the Pro to Leu mutation could disrupt activation since it is embedded in a sequence that makes direct contacts with the bound AdoMet. Deletion of isoleucine in the B12-binding domain would result in shortening of a beta-sheet. Our data provide the first evidence for mutations in the methionine synthase gene being culpable for the cblG phenotype. In addition, they suggest directly that mutations in methionine synthase can lead to elevated homocysteine, implicated both in neural tube defects and in cardiovascular diseases.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1996

Inversion polymorphism in natural populations of Drosophila bipectinata.

Inversion polymorphism was studied in seven natural populations of Drosophila bipectinata, five from northern and two from southern India. Chromosomal analysis of these populations revealed the presence of three paracentric inversions which are widespread in populations of D. bipectinata. Quantitative data indicated that the frequency of inversions and the level of inversion heterozygosity were very low in populations of D. bipectinata. There is no evidence for genetic differentiation between populations as a result of inversion polymorphism. These findings provide evidence for rigid chromosomal polymorphism in D. bipectinata. Significant non-random associations between inversions indicated epistatic interaction between inversions in natural populations of D. bipectinata.

Age Factors↗
PubMed1995

Structural and electronic similarity but functional difference in methylmalonyl-CoA mutase between coenzyme B12 and the analog 2',5'-dideoxyadenosylcobalamin.

The cofactor analog 2',5'-dideoxyadenosylcobalamin (ddAdoCbl) differs from the natural cofactor coenzyme B12 [5'-deoxyadenosylcobalamin (dAdoCbl)] by lacking only one oxygen atom. The 1H and 13C NMR spectra of ddAdoCbl have been assigned unambiguously by homonuclear and heteronuclear 2D NMR techniques. The 1H, 13C, and 31P chemical shift values for ddAdoCbl were compared with those of another organocobalamin, namely dAdoCbl. This assessment shows that the analog is very similar both electronically and structurally to the natural cofactor. The effectiveness of ddAdoCbl as a cofactor for both the human and Propionibacterium shermanii methylmalonyl-CoA mutases was compared with that of the natural cofactor. ddAdoCbl was found to be a competitive inhibitor with respect to dAdoCbl. Similar binding affinities to both enzymes were found for both the ddAdoCbl analog and the natural cofactor. However, in the presence of ddAdoCbl, the rate of conversion of methylmalonyl-CoA to succinyl-CoA was only 1-2% of that seen with the natural cofactor. There were no changes with time in the visible absorption spectrum of the bound cofactor analog in the presence of substrate, suggesting that the Co-C bond was not cleaved. The CD (circular dichroism) spectra of dAdoCbl and ddAdoCbl are very similar, consistent with the NMR results. The CD spectral changes upon binding to P. shermanii methylmalonyl-CoA mutase are large compared to those reported on the binding of dAdoCbl to ethanolamine ammonia lyase. Furthermore, the CD spectra of both enzyme-bound cobalamins are very similar, suggesting that similar changes in the conformation or structure in these cobalamins occur on binding to the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed1995

Demonstration that mammalian methionine synthases are predominantly cobalamin-loaded.

Methionine synthase is an important cellular housekeeping enzyme and is dependent on the cofactor cobalamin, a derivative of vitamin B12, for activity. It functions in two major metabolic pathways including the tetrahydrofolate-dependent one-carbon cycle and the salvage pathway for methionine. Its dysfunction has several physiological ramifications and leads to the development of megaloblastic anemia. In addition, it is suspected to be involved in the pathogenesis of neural tube defects. An issue that is central in weighing therapeutic options for methionine synthase-related disorders is the extent to which the enzyme exists as apoenzyme in vivo and, thus, can be potentially responsive to vitamin B12 therapy. despite the importance of this issue, the extent of holo- versus apoenzyme in mammalian tissue is controversial and unresolved. To address this question, we have developed a convenient anaerobic assay that employs titanium citrate to deliver low potential electron equivalents. The reductive activation of this enzyme is essential under in vitro assay conditions. We find that both the human placental and porcine liver methionine synthases exist predominantly in the holoenzyme form (90-100%) in the crude homogenate. In addition, the activity of the pure enzyme measured in the titanium citrate assay is also independent of exogenous cofactor, revealing that the cobalamin is tightly bound to the active site.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1995

Evidence from electron paramagnetic resonance spectroscopy of the participation of radical intermediates in the reaction catalyzed by methylmalonyl-coenzyme A mutase.

Recombinant methylmalonyl-coenzyme A (CoA) mutase from Propionibacterium shermanii has been purified 20-fold to near homogeneity in a highly active form. Neither the apoenzyme (the form in which the enzyme is isolated) nor the holoenzyme (reconstituted with the cofactor, adenosylcobalamin) has an electron paramagnetic resonance (EPR) spectrum associated with it. However, the addition of either the substrate, methylmalonyl-CoA, or the product, succinyl-CoA, results in the appearance of a transient EPR signal. The signal has hyperfine features that indicate coupling of the unpaired electron to the cobalt nucleus. In the presence of [CD3]methylmalonyl-CoA, an EPR signal is also seen and is similar to that obtained in the presence of protiated substrate. Power saturation studies reveal the presence of two components, a slow relaxing species (with an apparent g value of 2.11) and a fast relaxing species (with an apparent g value of 2.14) that can be partially resolved at low temperature and high power. The EPR-active intermediate is observed under catalytic conditions and is approximately midway in its resonance position between a free radical and cob(II)alamin. It is postulated to represent an exchange-coupled cob(II)alamin ... free radical pair. The signal bears close resemblance to those observed with partially dehydrated polycrystalline adenosylcobalamin following laser photolysis (Ghanekar, V.D., Lin, R.J., Coffman, R.E., and Blakley, R.L. (1981) Biochem. Biophys. Res. Commun. 101, 215-221) and with the adenosylcobalamin-dependent ribonucleotide reductase under freeze-quench conditions (Orme-Johnson, W.H., Beinert, H., and Blakley, R.L. (1974) J. Biol. Chem. 249, 2338-2343). When cob(II)alamin is generated under noncatalytic conditions (i.e. in the presence of propionyl-CoA or by electrochemical reduction of enzyme-bound hydroxocob-(III)alamin), a different EPR signal is observed with g = 2.26 and g = 2.00, typical of base-on cob(II)alamin.

Cobamides↗
PubMed1995

Chromosomal variability and interracial hybridization in Drosophila bipectinata.

Three paracentric inversions, In(D)2L, In(C)2R and In(H)3L, often persist in laboratory stocks of Drosophila bipectinata initiated from flies collected from natural populations. In order to test the effect of interracial hybridization on the degree of chromosomal variability, four stocks of D. bipectinata originating from different geographical localities and which were polymorphic due to the presence of three inversions, were crossed with each other. Hybrid lines as well as parental lines which served as controls were maintained in food bottles for ten generations. After ten generations, chromosomal analysis of hybrid lines as well as parental lines was undertaken. The results revealed that: all hybrid and parental lines remained polymorphic; there was a decline in the frequency of heterozygous inversions in certain hybrid populations of mixed geographical origin; and there was variation with respect to the degree of chromosomal variability in different crosses involving different inversions. These results are discussed in the light of findings reported in other Drosophila species.

Animals↗
PubMed1994

Inhibition of the human methylmalonyl-CoA mutase by various CoA-esters.

Human methylmalonyl-CoA mutase is inhibited by ethylmalonyl-CoA, cyclopropylcarbonyl-CoA carboxylate, and methylenecyclopropylacetyl-CoA, which are substrate, intermediate, and product analogs, respectively. The mode of inhibition by each analog is reversible and mixed with respect to the substrate, methylmalonyl-CoA. This implies that the inhibitors are able to bind to both free enzyme and to the enzyme-substrate complex, although with affinities that are 4.5- to 10-fold different for the two species. The Ki1 for the cyclopropylcarbonyl-CoA carboxylate (0.26 +/- 0.07 mM), is 4-fold greater than the Km(app) measured for the substrate, methylmalonyl-CoA. Additionally, ethylmalonyl-CoA functions as an alternate substrate and is metabolized to methylsuccinyl-CoA. The human mutase is a homodimer that binds 1 mol of cobalamin per subunit. So, the observed mixed inhibition kinetics by substrate analogs is curious. Our finding that methylenecyclopropylacetyl-CoA, the causative agent of Jamaican "vomiting sickness," inhibits methylmalonyl-CoA mutase, while interesting, is probably not physiologically important because of the relatively high inhibition constants (Ki1 = 0.47 +/- 0.12 mM and Ki2 = 2 +/- 0.34 mM) observed with this compound.

Acyl Coenzyme A↗
PubMed1994

Purification and kinetic mechanism of a mammalian methionine synthase from pig liver.

Porcine hepatic methionine synthase has been purified to near homogeneity. The enzyme is isolated in two forms which were purified approximately 9,000- and approximately 7,000-fold and were obtained in 0.9 and 2.5% overall yield, respectively. The mammalian enzyme from pig liver is a large monomeric protein with a molecular mass of 151-155 kDa. It is characterized by the absence of any metals other than cobalt which is associated with the cofactor, cobalamin. This enzyme, like the methionine synthase from Escherichia coli is dependent on S-adenosylmethionine for activity. The steady state kinetic studies demonstrate that the reaction operates via an ordered sequential mechanism in which binding of CH3-H4-folate precedes homocysteine, and methionine is released prior to H4-folate.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1994

Crystal structure of peanut lectin, a protein with an unusual quaternary structure.

The x-ray crystal structure of the tetrameric T-antigen-binding lectin from peanut, M(r) 110,000, has been determined by using the multiple isomorphous replacement method and refined to an R value of 0.218 for 22,155 reflections within the 10- to 2.95-A resolution range. Each subunit has essentially the same characteristic tertiary fold that is found in other legume lectins. The structure, however, exhibits an unusual quaternary arrangement of subunits. Unlike other well-characterized tetrameric proteins with identical subunits, peanut lectin has neither 222 (D2) nor fourfold (C4) symmetry. A noncrystallographic twofold axis relates two halves of the molecule. The two monomers in each half are related by a local twofold axis. The mutual disposition of the axes is such that they do not lead to a closed point group. Furthermore, the structure of peanut lectin demonstrates that differences in subunit arrangement in legume lectins could be due to factors intrinsic to the protein molecule and, contrary to earlier suggestions, are not necessarily caused by interactions involving covalently linked sugar. The structure provides a useful framework for exploring the structural basis and the functional implications of the variability in the subunit arrangement in legume lectins despite all of them having nearly the same subunit structure, and also for investigating the general problem of "open" quaternary assembly in oligomeric proteins.

Arachis↗
PubMed1994

Identification of a human immunodeficiency virus type 1 TAR binding protein in human hepatoblastoma HepG2 cells that trans-activates HIV-1 LTR-directed gene expression.

Recently, we have shown that the human immunodeficiency virus (HIV-1) long terminal repeat (LTR) directed chloramphenicol acetyltransferase (CAT) gene is efficiently expressed in human hepatoblastoma HepG2 cells and these cells can support productive HIV-1 replication. In this study we show that HepG2 cells contain a nuclear factor that binds to the HIV-1 trans-activating region (TAR), which we named HepG2-derived TAR binding protein (HTBP). Gel retardation assays using synthetic oligonucleotide probes carrying different mutations in the TAR region and competition DNA mobility-shift experiments using these oligonucleotides revealed the binding site encompassing between +7 and +13 nucleotides (5'-TCTGGTT-3') in the HIV-1 LTR. An in vivo CAT competition assay using -65HIV-1 LTR CAT as a reporter plasmid and various competitor plasmids containing these mutated oligonucleotides also demonstrated that HTBP can influence the HIV-1 LTR-directed CAT gene expression in HepG2 cells by interaction with a specific sequence in the TAR region.

Base Sequence↗