Distinctive Enzymes of Aromatic Amino Acid Biosynthesis That Are Highly Conserved in Land Plants Are Also Present in the Chlorophyte Alga Chlorella sorokiniana

Abstract

Considerable enzymological diversity underlies the capacity for biosynthesis of aromatic amino acids in nature. For this biochemical pathway, higher plants as a group exhibit a uniform pattern of pathway steps, compartmentation, and catalytic, physical and allosteric properties of enzymes. This biochemical pattern of higher plants contains a collection of features which are completely different from photosynthetic prokaryotes such as the cyanobacteria. A unicellular representative of the chlorophyte algae, Chlorella sorokiniana, was found to be strikingly similar to higher-plant plastids in possessing the following distinctive enzymes: a Mn 2+-stimulated, dithiothreitol-activated isoenzyme of 3-deoxy-D-araWno-heptulosonate 7-phosphate (DAHP) synthase, a probable bifunctional protein competent as both dehydroquinase and shikimate dehydrogenase, an allostericall y controlled isoenzyme of chorismate mutase, a highly thermotolcrant species of prephenate aminotransferase, an NADP+-dependent, tyrosine-inhibited arogenate dehydrogenase, and an arogenate dehydratase. In addition an isoenzyme of DAHP synthase shown in higher plants to be cytosolic, absolutely dependent upon the presence of divalent metals, and able to substitute other sugars for erythrose-4-phosphate, was also demonstrated in this alga. A broad-specificity 3-deoxy-D-ma/i no-octulosonate 8-phosphate synthase, recently discovered in higher plants, is also present in this Chlorella species. Keys words: L-Arogenate — Aromatic amino acid biosynthesis — Chlorella sorokiniana — Chlorophyte algae — Shikimate pathway. In higher plants an entire complement of enzymes performed by 3-deoxy-D-araW/io-heptulosonate 7-phoswhich specify aromatic amino acid biosynthesis is located phate (DAHP) synthase which catalyzes the condensawithin the plastid compartment (Jensen 1986). The com- tion of erythrose-4-p hosphate and phosphoenolpyruvate mon portion of the biosynthetic pathway consists of seven (PEP), dehydroquinate (DHQ) synthase, dehydroquinase, steps which thus far appear to be universal. These steps are an NADPH-utilizing shikimate (SHK) dehydrogenase, Abbreviations: ADH, arogenate dehydrogenase; ADT, arogenate dehydratase; AGN, L-arogenate; AroD-E, bifunctional protein exhibiting catalytic domains for dehydroquinase and shikimate dehydrogenase; AS, anthranilate synthase; CHA, chorismate; CM-1, plastid-localized isoenzyme of chorismate mutase in higher plants; CM-2, cytosolic isoenzyme of chorismate mutase; CM-O, species of chorismate mutase from Chlorella recovered in the gradient fractions following HA chromatography; CM-R, general designation for the class of chorismate mutase which includes CM-1, CM-W and CM-G and is monofunctional and subject to both allosteric activation by Trp and feedback inhibition by Tyr and Phe; CM-W, species of chorismate mutase from Chlorella recovered in the wash fractions following HA chromatogTaph y; DAHP, 3-deoxy-D-<waWno-heptulosonate 7-phosphate; DHQ, dehydroquinate; DS-Co, cytosolic isoenzyme of DAHP synthase; DS-Mn, plastid-localized isoenzyme of DAHP synthase; DTT, dithiothreitol; EPPS, N-(2-hydroxyethyl)piperazine-A/'-(3-propanesulfonic acid); HA, hydroxylapatite; KDOP, 3-deoxy-D-manno-octuIosonate-8-phosphate; OPA, orthopthalaldehyde; PDT, prephenate dehydratase; PEP, phosphoenolpyruvate; PIPES, 1,4-piperazinediethanesulfonic arid; PLP, pyridoxal-5'-phosphate; PMSF, phenylmethylsulfonyl fluoride; PPA, prephenate; PPY, phenylpyruvate; SHK, shikimate.