The reduction step in diaminopimelic acid biosynthesis.

In order to facilitate the elucidation of the remaining undetermined steps in diaminopimelic acid and lysine biosynthesis, mutants blocked in the pathway between aspartic semialdehyde and a-N-succinyl-e-ket.opimelic acid were sought. A mutant (M-203) with a requirement for diaminopimelate and lysine was obtained. An examination of the enzymatic composition of the extract prepared from the mutant, coupled with its nutritional requirements, indicated that it was blocked in the desired part of the biosynthetic pathway. It had previously been shown that crude extracts prepared from wild type Escherichia coli could synthesize diaminopimelic acid from its early precursors, aspartate and pyruvate (I). Similar extracts prepared from M-203 were deficient in the capacity to make diaminopimelic acid unless they were supplemented with an enzyme fraction from the wild type. This ability to restore the capacity for diaminopimelic acid synthesis to M-203 extracts was made the basis for an assay for the enzyme lacking in M-203. Although the substrate and product of t.he new enzyme were initially unknown, the enzyme could be extensively purified from wild type extracts with use of this assay. When the condensation step in diaminopimelate synthesis involving aspartic semialdehyde and pyruvate was discovered (2), it was shown that M-203 could carry out this reaction as well as wild type extracts. Later it was shown that the succinyl group of cu-N-succinyl-e-ketopimelic acid arose by a transfer from succinyl-Cob to LdQetrahydrodipicolinic acid (3). It was found that M-203 could also carry out this reaction. This left, as the most likely role for the enzyme that M-203 lacked, that of a reductase capable of converting the product of the condensing reaction to tetrahydrodipicolinic acid. This was verified by using the aspartic semialdehyde-pyruvate-condensing enzyme purified from M-203 to generate the substrate of the new enzyme. TPNH was added and remained in the reduced form until the new enzyme was also added, whereupon rapid oxidation of TPNH ensued. The enzyme was then purified with TPNH oxidation as the basis of a spectrophotometric assay. The parallelism in purification with both assays revealed that restoration and reductase activity were identical. This report deals with the discovery of the new enzyme, its partial purification from E. coli, its identification as dihydrodipicolinic acid reductase, and some of its properties. A preliminary communication of these findings has appeared (4).