| Abstract
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The uptake and metabolism of stereoisomers of amino acids were investigated in Fusobacterium nucleatum, an oral bacterium associated with oral infections, and Fusobacterium varium, an inhabitant of the gastrointestinal tract. F. nucleatum assimilated both isomers of glutamine, histidine and lysine, but only the scL-isomers of glutamic acid and serine. In F. varium, the scL-isomers of arginine and histidine were selectively taken up, along with both isomers of 3-aminobutyric acid, glutamic acid, lysine and serine. Although amino acids are major sources of energy for fusobacteria, the catabolism of scD-amino acids has not been investigated previously. The preferential uptake of scL-amino acids was employed to prepare gram quantities of scD-amino acids. Conditions were optimized for the preparation of scD-glutamate and scD-serine from their racemic mixtures by F. nucleatum. Recoveries of 70-80% were obtained, and the products had enantiomeric excesses >99%. Furthermore, it was demonstrated that scD-arginine and scD-histidine could be prepared under similar conditions by F. varium. The occurrence of three distinct pathways for the bacterial catabolism of glutamate to acetate, butyrate, NH3 and CO2 was investigated in F. nucleatum and F. varium using isotopically labelled substrates and enzyme assays. The acidic end-products (acetate and butyrate) were converted to p-bromophenacyl esters and separated prior to the determination of isotopic enrichments by nmr spectroscopy and mass spectrometry. The nonincorporation of label from scL- (5-C) glutamate and a major incorporation of C into C-1 of acetate and butyrate from scL- (1-C) glutamate indicated that the hydroxyglutarate pathway predominated and that participation of the aminobutyrate route was insignificant in F. nucleatum. The possible presence of the methylaspartate pathway was ruled out by the incorporation of label from scL- (4-C) glutamate into only C-2 of acetate and C-4 and C-2 of butyrate. Incorporation of (1,2-C2) - and usd\rm\lbrack\sp2H\sb3\rbrackusdacetate confirmed that the minor labelling of a second site in butyrate was due to the synthesis of butyrate from acetate produced by the hydroxyglutarate pathway. In F. varium, label was incorporated into C-1 of acetate and equally into C-1 and C-3 of butyrate from both scL- (1-C) - and scL- (4-C) glutamate, and unenriched carboxylic acids were isolated from the cultures supplemented with scL- (5-C) glutamate. Only the methylaspartate pathway is supported by these results, in contrast to an earlier suggestion that the three pathways operated simultaneously in F. varium. scD- (3-C) Glutamate was also catabolized by the methylaspartate pathway, ruling out differential metabolism of the glutamate enantiomers as the source of this discrepancy. In the cell-free extracts of F. varium, the metabolism of scD- and scL-glutamate and the formation of these amino acids from mesaconate was followed by hplc and nmr. A glutamate racemase was detected and partially purified from these extracts.
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