Biosynthesis of Aromatic Amino Acids by Isolated Spinach Chloroplasts — intracellular Compartmentation of the Reactions

Abstract

Chloroplasts have high capacities for reduction of NO− 2 to NH+ 4, for NH+ 4 assimilation, and for transamination of 2-oxoacids to amino acids. Still, the incorporation of 14CO2 into amino acids by isolated chloroplasts is very low, and probably due to contamination of other cellular material. Thus, it seems that the chloroplasts are dependent on other cellular compartments for the import of 2-oxoacids or precursors to 2-oxoacids. One possible exception from this ‘rule’ may be the biosynthesis of aromatic amino acids via the shikimic acid pathway. This pathway starts with the fusion of erythrose-4-phosphate and phosphoenolpyruvate (PEP) to eventually form shikimic acid, and after fusion with another molecule of PEP chorismic acid is formed; the latter is the common precursor to tyrosine, phenylalanine and tryptophan. Erythrose-4-phosphate is an intermediate in the Calvin cycle, and consequently available in the chloroplasts. PEP could be obtained from 3-phosphoglycerate (3-PGA), provided that the enzymes phosphoglycerate mutase and enolase are present in chloroplasts. This is, however, questionable. It has been shown (Bickel et al. 1979) that chloroplasts possess the enzymes of the shikimic acid pathway and may convert 14C-shikimic acid into aromatic amino acids. With unpurified chloroplasts also 14C from 14CO2 was incorporated. We have used purified chloroplasts in 14CO2 fixation experiments and reconstituted a possible cytoplasmic pathway from 3-PGA to PEP by adding PGA mutase and enolase to the incubation medium, If both enzymes were present during 14CO2 fixation the incorporation of 14C into aromatic amino acids was stimulated several fold. Thus, the results show that the PEP used by the chloroplasts in the shikimic acid pathway may be imported from the surrounding medium, i.e. the cytoplasm under in vivo conditions. The results further suggest that chloroplasts do not have PGA mutase or enolase activity.