On the utilization of D-fructose for pentose synthesis in Escherichia coli.

The major portion of the ribose and deoxyribose of the RNA and DNA of Escherichia coli does not arise from the oxidative pathway leading from hexose to pentose.1l3 At least 70 per cent of the pentose arises from nonoxidative pathways in which glucose is converted to triose by the Embden-Meyerhof pathway.4 Triose, in the form of glyceraldehyde 3-phosphate, then receives a two-carbon unit from a two-keto sugar, through the action of the enzyme transketolase. The operation of this pathway explains our findings that in the case where (1-018) glucose is used as the carbon source, all the 018 in the nucleosides isolated from the nucleic acids is in the 5'-positionA4 In a further study of this pathway, we have prepared (2-018) fructose and used it as the sole carbon source for the growth of E. coli. The oxidative pathway would not produce labeled ribosides, since the oxygen atom of the C-2 position of fructose would be converted to the oxygen atom at the C-1 position of ribose. The C-1 oxygen atom would then be lost in the formation of the sugar-base linkage of the nucleotide. In the nonoxidative pathway, we would expect that if the mechanism of action of aldolase in E. coli is the same as that of the aldolase in muscle,5' 6 all the 018 in the oxygen atom attached to the C-2 position of fructose would be lost in the formation of the Schiff base between fructose diphosphate and aldolase. Therefore, triose formed by the cleavage of fructose diphosphate would not contain 018. If the two-carbon unit received by the triose to form pentose is also derived from fructose, then it would be expected that there would be 018 in the 2'-position of the ribosides. The deoxyribosides, of course, would not contain 018 Analysis of the nucleosides did not confirm these expectations. Adenosine contained high concentrations of 018 in the 4'-position and lower concentrations in the 2'-position, while deoxyadenosine contained the same high concentration of label in the 4'-position. Apparently, the mechanism of action of aldolase in E. coli differs from that of aldolase in muscle.7 Materials and Methods.-(2-O18) Fructose: Ten gm of twice-recrystallized D-fructose were dissolved in 9 cc of 85 atom % excess H2018 (from Weizmann Institute of Science, Rehovoth, Israel) and incubated at 450 for 2 days. The water was removed by lyophilization. Alcohol was added to the syrupy residue, and the fructose crystallized. Analysis of the crystalline fructose showed 6.53 atom % excess 018 in the molecule (39.2 atom % excess 018 in the C-2 position). To verify the position of the 018 label, the 2,4-dinitrophenylhydrazone-pyridine solvate derivatives of the labeled fructose was prepared. Analysis of this compound showed 0.003 atom % excess 018. Procedure: E. coli B was grown in minimal medium9 at 370 on (2-018) fructose as the sole carbon source. The inoculating culture was previously grown on the labeled fructose. Fructose was added at the time of the inoculation to minimize the loss of 018 through spontaneous exchange with the medium.t The bacteria were harvested in early log