Abstract

The Escherichia coli genome codes for three highly homologous enzymes which use a 4Fe-4S mediated catalysis to deaminate L-serine and generate pyruvate and ammonia. The three enzymes are encoded by the genes sdaA, sdaB and tdcG . Enzymes of this type are found in many prokaryotes, and are not found in any eukaryotes. Despite the fact that the first E. coli L-serine deaminase (L-SD) was identified in 1955, and the regulation of its expression has been extensively studied, the physiological function of this delicately regulated enzyme is still unknown. In part one of this work, the impact of L-SD on the metabolism of E. coli K-12 was studied by creating a strain from which all three genes were deleted. This strain has serious growth problems. While the triple mutant grows well in glucose minimal medium even with L-serine, it forms long filaments on subculture into Luria Broth (LB). On subculture into minimal medium with glucose and casamino acids (CAA), it forms very large, abnormally shaped cells, many of which are osmotically sensitive and lyse. The processes of DNA replication and cell division are both abnormal in the triple mutant cell grown with CAA. Further study indicated that the triple mutant is unable to maintain sufficient production of one-carbon (C1) units. Provision of an exogenous supply of S-adenosylmethionine (SAM) restores cell division in LB, and repairs much of the difficulty in growth in medium with CAA. Whereas cells grown with CAA in the absence of SAM show abnormal FtsI production and localization within filaments, the addition of SAM produces filaments and normal recruitment of FtsI into the division septum. In part two, a mutant MEW128 which had been shown to be deficient in post-translational activation of L-SD was investigated. The mutation in that strain was located in ygfZ , a gene of unknown function. Neither MEW128 nor a strain known to carry a ygfZ deletion produces an active L-SD. Very little is known about the function of YgfZ, though it has been crystallized and shown by Teplyakov et al. to bind folates. The possibility that it is involved in assembly of the iron sulfur cluster (Fe-S) is considered.