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Query: UNIPROT:P06889 (Mol)
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The complex of Lactobacillus casei dihydrofolate reductase with trimethoprim and NADP+ exists in solution as a mixture of approximately equal amounts of two slowly interconverting conformational states [Gronenborn, A., Birdsall, B., Hyde, E. I., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1981) Mol. Pharmacol. 20, 145]. These have now been further characterized by multinuclear NMR experiments, and a partial structural model has been proposed. 1H NMR spectra at 500 MHz show that the environments of six of the seven histidine residues differ between the two conformations. The characteristic 1H and 31P chemical shifts of nuclei of the coenzyme in the two conformations of the complex are identical in analogous complexes formed with a number of trimethoprim analogues, indicating that the nature of the two conformations is the same in each case. The pyrophosphate 31P resonances have been assigned to the two conformations, and integration of the 31P spectrum shows that the ratio of conformation I to conformation II varies from 0.4 to 2.3 in the complexes with the various trimethoprim analogues, the ratio for the trimethoprim complex itself being 1.2. Transferred NOE experiments, together with the 1H and 13C chemical shifts, indicate that in conformation II of the complex the nicotinamide ring of the coenzyme has swung away from the enzyme surface into solution; this is made possible by changes in the conformation of the pyrophosphate moiety. In conformation I, by contrast, the nicotinamide ring remains bound to the enzyme. 13C and 15N experiments show that trimethoprim is protonated on N1 in both conformations of the ternary complex. Analysis of the 1H chemical shifts of trimethoprim in terms of ring current effects shows that in conformation I of the ternary complex trimethoprim retains the same conformation as in its binary complex, but 13C, 15N, and 19F [using 2,4-diamino-5-(3,5-dimethoxy-4-fluoro-benzyl)pyrimidine] experiments show that the environment of both the pyrimidine ring and benzyl ring is affected by the proximity of the coenzyme. Less information is available about the conformation of the inhibitor in conformation II of the complex, but its environment is similar to that in the binary enzyme-inhibitor complex. The implications of the existence of these two conformations of the enzyme for understanding cooperativity in binding between NADP+ and trimethoprim are briefly discussed.
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PMID:Multinuclear NMR characterization of two coexisting conformational states of the Lactobacillus casei dihydrofolate reductase-trimethoprim-NADP+ complex. 643 42

Tissue levels of NAD+ appear to be regulated primarily by the concentration of extracellular nicotinamide, which in turn is controlled by the liver in a hormone-sensitive manner. Hepatic regulation involves the conversion of excess serum nicotinamide to 'Storage NAD+' and inactive excretory products, and the replenishment of serum nicotinamide by the hydrolysis of 'Storage NAD+.' Tryptophan and nicotinic acid contribute to 'Storage NAD+,' and thus are additional sources of nicotinamide. In response to administered nicotinamide, there is a preferential utilization of ATP and PRPP (5-phosphorylribose-1-pyrophosphate) for the biosynthesis of NAD+. This biosynthetic priority, whose purpose appears to be the conservation of intracellular nicotinamide, may explain why nicotinamide inhibits RNA and DNA synthesis in regenerating tissues and why elevated nicotinamide levels are toxic to growing animals and to mammalian cells in culture.
Mol Cell Biochem 1980 Dec 16
PMID:Physiology aspects of pyridine nucleotide regulation in mammals. 645 Aug 78

In order to address the problem of amino acid auxotroph scarcities in photosynthetic organisms, an attempt was made to recover methionine auxotrophs in a unicellular green alga, Chlamydomonas reinhardtii. Evidence of methionine permeation into the algal cells was provided by the successful competition of the amino acid with its antimetabolite, methionine sulfoximine. No methionine auxotrophs were isolated despite the frequent recovery of both nicotinamide and arginine auxotrophs, selected as controls, and of methionine sensitive mutants. The use of either N-methyl-N'-nitro-N-nitrosoguanidine or ultraviolet light as mutagens appeared not to alter the mutation spectrum. Medium containing methionine was shown to be inhibitory to the growth of cells in culture under fluorescent light and this toxicity was further stimulated in the presence of riboflavin. In view of our results, and the observations of other studies, the non-recoverability of methionine auxotrophs is discussed as a function of the photodynamic action of methionine.
Mol Gen Genet 1981
PMID:Is it possible to isolate methionine auxotrophs in Chlamydomonas reinhardtii? Consideration of photodynamic action of the amino acid. 645 54

Glyceraldehyde 3-phosphate dehydrogenase is a tetramer of four chemically identical subunits which requires the cofactor nicotinamide adenine dinucleotide (NAD) for activity. The structure of the holo-enzyme from Bacillus stearothermophilus has recently been refined using X-ray data to 2.4 A resolution. This has facilitated the structure determination of both the apo-enzyme and the enzyme with one molecule of NAD bound to the tetramer. These structures have been refined at 4 A resolution using the constrained-restrained parameter structure factor least-squares refinement program CORELS. When combined with individual atomic temperature factors from the holo-enzyme, these refined models give crystallographic R factors of 30.2% and 30.4%, respectively, for data to 3 A resolution. The apo-enzyme has 222 molecular symmetry, and the subunit structure is related to that of the holo-enzyme by an approximate rigid-body rotation of the coenzyme binding domain by 4.3 degrees with respect to the catalytic domains, which form the core of the tetramer. The effect of this rotation is to shield the coenzyme and active site from solvent in the holo-enzyme. In addition to the rigid-body rotation, there is a rearrangement of several residues involved in NAD binding. The structure of the 1 NAD enzyme is asymmetric. The subunit which contains the bound NAD adopts a conformation very similar to that of a holo-enzyme subunit, while the other three unliganded subunits are very similar to the apo-enzyme conformation. This result provides unambiguous evidence for ligand-induced sequential conformational changes in B. stearothermophilus glyceraldehyde 3-phosphate dehydrogenase.
J Mol Biol 1984 Sep 25
PMID:Structural evidence for ligand-induced sequential conformational changes in glyceraldehyde 3-phosphate dehydrogenase. 649 62

To uncover the functional circuitry both within the mitochondrial genome and between the mitochondrial and the nuclear genome, we have developed a general method for selecting and characterizing genetically suppressor mutations that restore the respiratory capacity of mit- mitochondrial mutants. Several hundreds of pseudo-wild type revertants due to a second unlinked mutation which suppresses a target mit- mutation were isolated. The suppressor mutations were found located either in the nuclear (abbreviated NAM for 'nuclear accommodation of mitochondria') or in the mitochondrial genome (abbreviated MIM for 'mitochondrial-mitochondrial interaction'). The specificity of action of various suppressors upon some 250 different mit- mutations located in several genes was tested. According to this specificity of action, suppressors were subdivided into two major classes: allele specific or gene specific suppressors. Because the cob-box mitochondrial gene has a mosaic organization, we were able to find a novel third class of extragenic suppressors specific for mit- mutations within the introns of this gene. Four examples of suppressors showing various specificities of action illustrate our approach. (1) a nuclear gene controlling specific alleles of different mitochondrial genes; (2) a nuclear gene controlling selectively one intron of a split mitochondrial gene; (3) a mitochondrial gene controlling specific alleles of different mitochondrial genes; (4) a region in one complex mitochondrial gene which controls selectively one intron of another split mitochondrial gene. Different mechanisms of suppression are discussed stressing the alleviation of splicing deficiencies of intron mutations.
Mol Gen Genet 1980
PMID:Long range control circuits within mitochondria and between nucleus and mitochondria. I. Methodology and phenomenology of suppressors. 700 99

Particles carrying heterogeneous nuclear RNA (30--40 S-particles) were isolated from rat liver nuclei and the particle proteins separated by sodium dodecylsulfate gel electrophoresis. Some properties of a 110000 molecular weight component (P 110/103) were studied in detail: (i) P 110/103 was labeled to a 4-5 times higher specific activity than the major particle proteins in the presence of [14C]-amino acids in vivo. (ii) In nuclei incubated with [3H]- or [32P]-nicotinamide adenine dinucleotide P 110/103 was labeled presumably by ADP-ribosylation. (iii) A protein with the same molecular weight as P 110/103 and isolated from the nuclear extract by affinity chromatography was phosphorylated in vitro.
Mol Biol Rep 1981 May 22
PMID:Properties of a 110000 molecular weight protein in rat liver hnRNP particles. 725 6

L-2-Hydroxyisocaproate dehydrogenase (L-HicDH) from Lactobacillus confusus, a homotetramer with a molecular mass of 33 kDa per subunit, belongs to the protein family of the NAD(+)-dependent L-2-hydroxycarboxylate dehydrogenases. L-HicDH was crystallized with ammonium sulphate as precipitant in the presence of NAD+. The crystals belong to the trigonal space group P3(2)21, with a = 135.9 A and c = 205.9 A, and diffract X-rays to 2.2 A resolution. The crystal structure was solved by Patterson search and molecular replacement techniques and refined to an R-value of 21.4% (2.2 to 8 A). The final structure model contains one NAD+ molecule and one sulphate ion per subunit, with 309 water molecules. An unusual feature of this crystal structure is the deviation of the protein subunits from non-crystallographic symmetry, which is so strong that it can be detected globally by self-rotation calculations in reciprocal space. This asymmetry is especially pronounced in the environment of the active site; it is reflected also in the nicotinamide conformation of NAD+ and allows some conclusions to be drawn about the catalytic mechanism. In this context, an "inner active site loop" is identified as a structural element of fundamental functional importance. Furthermore, with knowledge of the crystal structure of L-HicDH the differences in substrate specificity between L-HicDH and the L-lactate dehydrogenases can be partly explained.
J Mol Biol 1995 Aug 11
PMID:Crystal structure of L-2-hydroxyisocaproate dehydrogenase from Lactobacillus confusus at 2.2 A resolution. An example of strong asymmetry between subunits. 764 2

The combination method of carboxypeptidase Y digestion and fast atom bombardment (FAB) mass spectrometry is described for the identification of C-terminal amino acid amides in peptides. Carboxypeptidase Y has amidase activity as well as exopeptidase activity in the same digestion buffer condition. Based on this concept, we develop a new technique which can definitively and easily identify the C-terminal amino acid amides. This method obviates the need for several complicated steps occurring in previous methods, but improves sensitivity, and enables exact identification of the amino acid amide by the difference of molecular mass. Analyses of carboxypeptidase Y digested peptides, not liberated free amino acid amides, were carried out by fast atom bombardment mass spectrometry. The use of truncated peptides by fast atom bombardment mass spectrometry in C-terminal amino acid amide determination gives several advantages over analyses of the liberated amino acid amides. The C-terminal amino acid amides of Allantostatin I (Leu-NH2), alpha-Melanocyte Stimulating Hormone (Val-NH2), and Ranatensin (Met-NH2) are unequivocally determined at a level of 0.90-2.3 nmol per peptide. This approach is based on entirely different principles than the previous approaches.
Biochem Mol Biol Int 1994 Nov
PMID:Identification of the C-terminal amino acid amides by carboxypeptidase Y digestion and fast atom bombardment mass spectrometry. 770 6

The refined crystal structures of the recombinant cytochrome b reductase fragment of corn (Zea mays) nitrate reductase, its ADP complex and the active-site mutant Cys242Ser are reported here. The native structure has been refined at 2.5 A resolution to a crystallographic R-factor of 18.7% with root-mean-square (r.m.s) deviations from standard bond lengths and angles of 0.013 A and 2.0 degrees. The diffraction pattern of the crystals is highly anisotropic and correction of this effect lowered the crystallographic R-factor by 5% during the refinement. The structure of the enzyme co-crystallized with ADP has been solved at 2.7 A resolution and refined to an R-factor of 18.6% with r.m.s. deviations from standard bond lengths and angles of 0.014 A and 2.1 degrees. It revealed the binding site of the ADP moiety of the NADH cofactor, which is the electron donor for nitrate reduction. Based on this structure, a model of NADH at the active site of the enzyme was built and the implications for electron transfer from NADH to the flavin cofactor are discussed. The crystal structure of an active-site mutant enzyme, Cys242Ser, has been solved by difference Fourier synthesis and refined to an R-factor of 19.0% to 3.0 A resolution with standard deviations of bond lengths and angles of 0.017 A and 2.5 degrees. This structure analysis suggests that the observed decrease in catalytic activity of this mutant might be due to misalignment of the nicotinamide ring in its binding site. A model of the heme-containing domain of nitrate reductase has been built based on the X-ray structure of bovine cytochrome b5 and has been docked with the cytochrome b reductase fragment of nitrate reductase. The model of the complex contains six salt-bridges at the domain-domain interface and a hydrophobic core. In this model, His48, an invariant residue in the cytochrome b reductase family, forms an interaction with the propionic acid group of the D-ring of the heme cofactor. This group is in contact with the C-8 methyl group of the flavin ring. Residues that might influence the redox potential of the flavin cofactor are proposed and their possible role in electron transfer is discussed.
J Mol Biol 1995 May 19
PMID:Structural studies on corn nitrate reductase: refined structure of the cytochrome b reductase fragment at 2.5 A, its ADP complex and an active-site mutant and modeling of the cytochrome b domain. 776 Mar 34

The amino acid sequences of several bacterial toxin ADP-ribosyltransferases, rabbit skeletal muscle transferases, and RT6.2, a rat T-cell NAD glycohydrolase, contain three separate regions of similarity, which can be aligned. Region I contains a critical histidine or arginine residue, region II, a group of closely spaced aromatic amino acids, and region III, an active-site glutamate which is at times seen as part of an acidic amino acid-rich sequence. In some of the bacterial ADP-ribosyltransferases, the nicotinamide moiety of NAD has been photo-crosslinked to this glutamate, consistent with its position in the active site. The similarities within these three regions, despite an absence of overall sequence similarity among the several transferases, are consistent with a common structure involved in NAD binding and ADP-ribose transfer.
Mol Cell Biochem 1994 Sep
PMID:Common structure of the catalytic sites of mammalian and bacterial toxin ADP-ribosyltransferases. 789 62


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