Please use this identifier to cite or link to this item:
|Title:||The assimilation of L-threonine by an Arthrobacter sp.|
|Presented at:||University of Leicester|
|Abstract:||An Arthrobacter sp. which is capable of utilising L-threonine as the sole source of carbon and nitrogen for growth, has been isolated from soil by elective culture. The metabolic pathway by which this organism converts L-threonine into more common intermediary metabolites has been elucidated, and the properties of two key enzymes of the pathway have been studied. L-Threonine was shown to be oxidised in an NAD-dependent reaction catalysed by an inducible L-threonine dehydrogenase. Incorporation studies, in which [U-14c] L-threonine was added to the medium of organisms growing at the expense of threonine, or incubated with cell-free extracts of such organisms, indicated that in the Arthrobacter sp. the decarboxylation of AOB was of no physiological importance. Results showed that AOB was instead cleaved in a CoA-dependent reaction to yield acetyl-CoA and glycine. The enzyme which catalysed this reaction, aminoacetone synthase, was detected in cell-free extracts of threonine-grown organisms, and was inducibly formed only during growth on L-threonine; in particular it was not present in cells grown on acetate plus glycine. The cleavage reaction was reversible, and the activity of aminoacetone synthase was routinely assayed by the condensation of acetyl-CoA and glycine, and the formation of aminoacetone via AOB. Further incorporation studies, using [2-14c] acetate and [2-14c] glycine, showed that the acetyl-CoA and glycine derived from threonine were metabolised via the tricarboxylic acid plus glyoxylate cycles, and via the "folate-dependent "serine pathway" respectively (Sagers and Gunsalus, 1961). Extracts of threonine-grown organisms were shown to be rich in two key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, and in the component enzymes of the "serine pathway", viz. the "glycine cleavage system", L- serine hydroxymethyltransferase and L-serine dehydratase. L-Threonine dehydrogenase and aminoacetone synthase were partially purified from crude extracts of threonine-grown organisms, in order that their properties could be investigated. Certain preparations of L-threonine dehydrogenase were completely dependent upon the provision of bivalent cations for enzyme activity - this requirement being best fulfilled by Zn2+. Inhibitor studies confirmed the dependence upon Zn2+, and showed that the enzyme possessed one or more free thiol groupes which were essential for enzyme activity. The results of studies with purified aminoacetone synthase suggested that the enzyme was dependent upon pyridoxal 5'-phosphate for activity. By using the two purified enzymes it was convincingly demonstrated that both were required for the conversion of threonine into glycine and acetyl-CoA. The collaborative and sequential action of L-threonine dehydrogenase and aminoacetone synthase provides a novel route for the catabolism of threonine. This route differs fundamentally from that postulated by Elliott (1959, 1960), in which L-threonine is oxidised to aminoacetone, and this in turn metabolised to pyruvate via methylglyoxal and D-lactate. The pathway operating in the Arthrobacter sp. also provides a key metabolic role for aminoacetone synthase, an enzyme which had previously been thought to function solely in a somewhat speculative cyclical route for the oxidation of glycine (Elliott, 1959, 1960), by catalysing the production of aminoacetone from acetyl-CoA and glycine. A mechanism for the concerted action of L-threonine dehydrogenase and aminoacetone synthase which does not involve the production of free AOB is proposed and discussed. (Abstract shortened by UMI.).|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Biochemistry|
Items in LRA are protected by copyright, with all rights reserved, unless otherwise indicated.