Normal mode refinement of anisotropic thermal parameters for a supramolecular complex at 3.42-A crystallographic resolution.
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Here we report a normal-mode-based protocol for modeling anisotropic thermal motions of proteins in x-ray crystallographic refinement. The foundation for this protocol is a recently developed elastic normal mode analysis that produces much more accurate eigenvectors without the tip effect. The effectiveness of the procedure is demonstrated on the refinement of a 3.42-A structure of formiminotransferase cyclodeaminase, a 0.5-MDa homooctameric enzyme. Using an order of magnitude fewer adjustable thermal parameters than the conventional isotropic refinement, this protocol resulted in a decrease of the values of R(cryst) and R(free) and improvements of the density map. Several poorly resolved regions in the original isotropically refined structure became clearer so that missing side chains were fitted easily and mistraced backbone was corrected. Moreover, the distribution of anisotropic thermal ellipsoids revealed functionally important structure flexibility. This normal-mode-based refinement is an effective way of describing anisotropic thermal motions in x-ray structures and is particularly attractive for the refinement of very large and flexible supramolecular complexes at moderate resolutions. (+info)
Moonlighting glutamate formiminotransferases can functionally replace 5-formyltetrahydrofolate cycloligase.
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Channeling between the active sites of formiminotransferase-cyclodeaminase. Binding and kinetic studies.
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Formiminotransferase-cyclodeaminase, a circular tetramer of dimers, binds four tetrahydropteroylpolyglutamates/octamer, which indicates that these polyglutamate sites are formed by one type of subunit interface. The transferase and deaminase are separate catalytic sites as determined by inhibition studies with (6R)-tetrahydropteroylglutamate and by the observation that the activities can operate simultaneously. Under conditions where the transferase is saturated with tetrahydropteroyl(glutamate)n substrate, exogenously added formimino intermediate is utilized by the deaminase only if at least one of the substrate/intermediate pair is a monoglutamate. These properties indicate the existence of only one polyglutamate site/pair of catalytic sites. Kinetic specificity for each activity as measured by Vm/Km increases for longer polyglutamates, but does not differentiate among 4, 5, 6, and 7 glutamates. The enzyme shows distinct preference for hexaglutamate based on Kd as well as on Km values. With all substrates, Vm of the deaminase is greater than that of the transferase, allowing for potential channeling of the intermediate between active sites. Efficiency of channeling, optimal with pentaglutamate, does not correspond with affinity for binding. This demonstrates that a steric requirement predominates over simple sequestering of intermediates on the enzyme surface as the fundamental mechanism for channeling. (+info)
Enhancement of histidine and one-carbon metabolism in rats fed high levels of retinol.
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Histidine metabolism was studied in rats fed 10% casein diets supplemented with 1000 IU of retinol/g concurrent with or previous to exposure to high levels of dietary histidine (1% or 2%). When a retinol-supplemented 10% casein + 1% histidine diet was fed ad libitum for 21 days, urinary excretion of formiminoglutamic acid (FIGLU) was decreased by 50-70% over the entire period and plasma histidine was reduced by 30-70% for 16 days compared to rats receiving 10% casein + 1% histidine with normal levels of retinol. Rats pretreated for 10 days with a 10% casein diet supplemented with high levels of retinol oxidized 30% more L-[ring-2-14C]histidine to 14CO2 and excreted 76% less of the administered dose as urinary FIGLU compared to control rats not pretreated with high levels of retinol. Depression in growth due to supplementation of a 10% casein diet with 1% histidine were also partially alleviated in rats that were first pretreated with retinol. Activities of histidase, urocanase, and formiminoglutamic acid formiminotransferase (FIGLU transferase) were unaffected by retinol supplementation. The results suggest that retinol supplementation enhances histidine catabolism by exerting a change on one-carbon metabolism. (+info)
Methionine synthesis, aminoimidazole carboxamide excretion and folate levels in pregnant rats.
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The capacity for tetrahydrofolate regeneration through folate-linked methionine synthesis and for purine-ring closure through formylation of aminoimidazole carboxamide ribotide was studied in pregnant female rats fed diets containing either methionine or homocystine with or without folic acid. Plasma and liver folates, serine transhydroxymethylase, 5,10-methylene tetrahydrofolate dehydrogenase and glutamate formiminotransferase activities were also assayed. Pregnancy proceeded normally in all groups. Hypotrophic fetuses were observed only with the diet containing homocystine and no folic acid. Plasma folates were severely depleted at the end of pregnancy even when folic acid was present in the diet. Hepatic stores of folate were twice as high in the methionine as in the homocystine-fed pregnant females supplemented with folic acid. This favorable effect of methionine was not observed in folic acid-deficient females. No change in levels of serine transhydroxymethylase, 5,10-methylenetetrahydrofolate dehydrogenase, glutamate formimino-transferase activities was observed. Pregnancy did not stimulate methionine synthetase activity, the level of which was primarily affected by the nutritional conditions. Because of its low output and narrow range of adaptativity, methionine synthetase cannot be the sole regulatory factor of THF regeneration. Urinary excretion of aminoimidazole carboxamide was enhanced in folic acid-deficient pregnant females and was not prevented by supplying methionine. (+info)
A formiminotransferase cyclodeaminase isoform is localized to the Golgi complex and can mediate interaction of trans-Golgi network-derived vesicles with microtubules.
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A protein of 60 kDa (p60) has been identified using a quantitative in vitro vesicle-microtubule binding assay. Purified p60 induces co-sedimentation with microtubules of trans-Golgi network-derived vesicles isolated from polarized, perforated Madin-Darby canine kidney cells. Sequencing of the cDNA coding for this protein revealed that it is the chicken homologue of formiminotransferase cyclodeaminase (FTCD), a liver-specific enzyme involved in the histidine degradation pathway. Purified p60 from chicken liver has formiminotransferase activity, confirming that it is FTCD or an isoform of this enzyme. Isoforms of FTCD were identified in chicken hepatoma and HeLa cells, and immunolocalize to the region of the Golgi complex and vesicular structures in its vicinity. Furthermore, 58K, a previously identified microtubule-binding Golgi protein from rat liver (Bloom, G. S., and Brashear, T. A. (1989) J. Biol. Chem. 264, 16083-16092), is identical to FTCD. Both proteins co-purify with microtubules and co-localize with membranes of the Golgi complex. The capacity of FTCD to bind both to microtubules and Golgi-derived membranes may suggest that this protein, or one of its isoforms, might have in addition to its enzymatic activity, a second physiological function in mediating interaction of Golgi-derived membranes with microtubules. (+info)