Transport of Biosynthetic Intermediates: Homoserine and Threonine Uptake in Escherichia coli

Kinetics of the transport systems common for entry of L-isoleucine, L-leucine, and L-valine in Salmonella typhimurium LT2 have been analyzed as a function of substrate concentration in the range of 0.5 to 45 ,uM. The systems of transport mutants, KA203 (ilvT3) and KA204 (ilvT4), are composed of two components; apparent Km values for uptake of isoleucine, leucine, and valine by the low Km component are 2 ,uM, 2 to 3 uM, and 1 uM, respectively, and by the high Km component 30 ,uM, 20 to 40 uM, and 0.1 mM, respectively. The transport system(s) of the wild type has not been separated into components but rather displays single Km values of 9 ,uM for isoleucine, 10 4M for leucine, and 30 ,uM for valine. The transport activity of the wild type was repressed by L-leucine, aketoisocaproate, glycyl-L-isoleucine, glycyl-L-leucine, and glycyl-L-methionine.

mentioning

confidence: 99%

“…The transport activity of cells is repressible by certain amino acids, especially L-leucine (3,4,7,9,10,15,17,19,20,22,23). The entry of branched-chain amino acids into cells is inhibited by the presence of several amino acids in the medium (7,8,12,16,20,22,24).…”

mentioning

confidence: 99%

Repression and inhibition of transport systems for branched-chain amino acids in Salmonella typhimurium

Kiritani¹,

Ohnishi²

1977

“…In contrast, the regulation of branched-chain amino acid transport has received much less attention. A transport system for leucine, isoleucine, and valine is repressible by leucine but not isoleucine and valine (39,41), although other studies have implicated the latter two amino acids (50-51) as well as cysteine (27), methionine, and alanine (21)(22)(50)(51). A regulatory system that involves both cognate (leucine, isoleucine, and valine) and noncognate (cysteine, methionine, and alanine) amino acids would be very unusual and seemed unduly complex.…”

mentioning

confidence: 99%

Regulation of branched-chain amino acid transport in Escherichia coli

Quay¹,

Oxender²

1976

The repression and derepression of leucine, isoleucine, and valine transport in Escherichia coli K-12 was examined by using strains auxotrophic for leucine, isoleucine, valine, and methionine. In experiments designed to limit each of these amino acids separately, we demonstrate that leucine limitation alone derepressed the leucine-binding protein, the high-affinity branched-chain amino acid transport system (LIV-I), and the membrane-bound, low-affinity system (LIV-II). This regulation did not seem to involve inactivation of transport components, but represented an increase in the differential rate of synthesis of transport components relative to total cellular proteins. The apparent regulation of transport by isoleucine, valine, and methionine reported elsewhere was shown to require an intact leucine biosynthetic operon and to result from changes in the level of leucine biosynthetic enzymes. A functional leucyltransfer ribonucleic acid synthetase was also required for repression of transport. Transport regulation was shown to be essentially independent of ilvA or its gene product, threonine deaminase. The central role of leucine or its derivatives in cellular metabolism in general is discussed. filter (Nalgene filter unit, Nalge Sybron Corp., Rochester, N.Y.). Distilled water was sterilized by autoclave. Cultures (50 ml) for enzyme or transport assays were grown aerobically in 250-ml Erlenmeyer flasks in a shaking water bath (New Brunswick Scientific Co., model G-76) that maintained a constant temperature between 30 and 41 + 0.250C. The platform rotation was approximately 150 rpm. For the isola-1225

“…3) or 30 s. It is apparent from the data presented below that the Kt determinations include the equilibrium between methionine in transport and formation of SAM via the SAM synthetase reaction. A lowaffinity transport system also was identified with a K, of approximately 2 x 10-4 M. The latter system may be comparable to the isoleucine-valine-threonine system of Templeton and Savageau (23).…”

Section: Resultsmentioning

confidence: 69%

Methionine transport in Yersinia pestis

Montie¹,

Montie²

1975

Yersinia pestis TJW, an avirulent wild-type strain, requires phenylalanine and methionine for growth. It was of interest to examine and define the methionine transport system because of this requirement. The methionine system showed saturation kinetics with a Km for transport of approximately 9 x 10-' M. After 8 s of methionine transport, essentially all of the methionine label appeared in S-adenosyl-L-methionine (SAM) as detected in ethanol extracts. Small amounts of free methionine was detected intracellularly after 1 min of transport. Addition of glucose increased significantly the amount of intracellular methionine at 1 min. A series of SAM metabolic products was detected after 90 s to 5 min of transport including: 5'-thiomethyladenosine, homoserine lactone, S-adenosyl homoserine, and a fluorescent methyl receptor compound. Results from assays for SAM synthetase in spheroplast fractions showed a small (16%) but significant portion of synthetase associated with the membrane. However, most of the enzyme activity was associated with the cytoplasmic fraction. Methionine transport was characterized by a high degree of stereospecificity. No competition occurred from structurally unrelated amino acids. Although uptake was inhibited by uncoupling and sulfhydryl reagents, no efflux was observed. Results using energy inhibitors on unstarved and starved cells showed that respiratory inhibitors such as potassium cyanide (KCN) and amytal were most effective, and that arsenate was least effective. KCN plus arsenate completely blocked utilization of energy derived from glucose, and KCN completely blocked utilization of energy deived from D-lactate. The data indicate that methionine transport in Y. pestis is linked to the trapping of methionine in SAM. The results further suggest that this transport system can be classified as a permease-bound system where transport is coupled to an energized membrane state and to respiration.