The elucidation of the microbial pathways for amino acid biosynthesis in Escherichia coli and Neurospora crassa developed from the analysis of mutants, radioisotope studies, and then extensive enzymatic investigations (1). However, the intermediate biosynthetic steps in E. coli are now known to differ from the intermediates for the biogenesis of: serine in various biological systems (1); for lysine in higher fungi (2); for glutamate in Acetobacter suboxydans (3) and Clostridium kluyveri (4, 5); for alanine in C. kluyveri (6) and C. pasteurianum (7); and for cysteine in Aspergillus nidulans [cf. (8)]. With respect to valine, isoleucine, and leucine formation, Ruminococcus flavefaciens and several other rumen anaerobes are different from E. coli in their reliance on a reductive carboxylation of a branched chain fatty acid and animation to produce the corresponding amino acid [(9, 10) and earlier references cited therein]. Thus the a priori transfer of a biosynthetic pathway from one organism to another must be replaced by direct experimental inquiry to determine whether biological unity or diversity prevails.
To screen various biological systems for a specified pathway, investigators have used the incorporation of radioactive aspartate into lysine (2) as a marker of the routes of lysine biosynthesis. Similarly the demonstration of 5-dehydroquinase (11) and shikimic acid kinase (12) were initial clues of the path of biosynthesis of aromatic amino acids in a variety of microorganisms or plants. The presence of dihydroxyacid dehydratase (2, 3-dihydroxyacid hydrolyase, E.C.4.2.1.9.) was employed as one of several indicators of a specific sequence in valine-isoleucine biosynthesis with glucose-grown bacteria (13, 14), higher fungi (15), and higher plants (16, 17). The selection of the dehydratase was based on its unique substrates (13, 17) and its catalysis of the conversion of α,β-dihydroxyisovalerate to α-ketoiso valerate, and α,β-dihydroxy-β-methyl-n-valerate to α-keto-β-methyl-n-valerate, which are intermediates in valine and isoleucine formation.
