GENETIC CHARACTERIZATION OF A HIGHLY EFFICIENT ALTERNATE PATHWAY OF SERINE BIOSYNTHESIS IN ESCHERICHIA COLI (TUT CYCLE, THREONINE DEHYDROGENASE, PSEUDOREVERTANTS

PAULA DENISE RAVNIKAR, Purdue University

Abstract

There exists in Escherichia coli a group of enzymes that can function in an efficient and concerted way to convert threonine to serine. This sequence of reactions, designated the Tut (threonine utilization) Cycle, proceeds as follows: (a) threonine is oxidized, then cleaved to glycine and AcCoA; (b) glycine is converted to CO(,2), NH(,3) and a one carbon unit; (c) the resultant one carbon unit undergoes condensation with a second mole of glycine to yield serine; (d) the AcCoA formed in step (a) is recycled into the C4 pool of metabolic intermediates via the glyoxylate cycle. The evidence for the operation of the Tut Cycle is as follows: Strains of E. coli having lesions in serA, B, C or glyA grow rapidly and without appreciable lag on minimal media supplemented with elevated levels of leucine, arginine, lysine, threonine, and methionine. The growth of ser mutants in this media does not occur if the strain has an additional lesion in glyA (serine hydroxymethyl-transferase), gcv (the glycine cleavage system) or tdh (threonine dehydrogenase). Pseudorevertants of ser mutants capable of growth either on unsupplemented minimal media or media supplemented with low levels of leucine, arginine, lysine, threonine and methionine have been isolated. At least two unlinked mutations are associated with such phenotypes. Pseudorevertants into which lesions in glyA, gcv or tdh were introduced grew only on serine or glycine supplemented media. In addition, a regulatory role is postulated for the thrA gene product when pseudorevertants are growing on minimal media. I have characterized a plasmid, pDR121, that contains the tdh gene. The tdh gene has been found to lie in the 83 minute region of the E. coli chromosome. Threonine dehydrogenase levels rise when ser mutants utilize the Tut Cycle for serine biosynthesis. Furthermore, multicopy plasmids bearing the tdh or tdh and thrjA genes alleviate the nutritional requirements of serA, B, C, and glyA mutants. I propose that the principal physiological role of threonine dehydrogenase in E. coli is to function as the first step in a series of reactions that drew upon the C4 pool of metabolic intermediates, rather than the C3 pool, for serine biosynthesis.

Degree

Ph.D.

Subject Area

Molecular biology

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