Functional rescue of a bacterial dapA auxotroph with a plant cDNA library selects for mutant clones encoding a feedback-insensitive dihydrodipicolinate synthase

Dihydrodipicolinate synthase (DHDPS; EC4.2.1.52) catalyses the first reacti on of lysine biosynthesis in plants and bacteria. Plant DHDPS enzymes are s trongly inhibited by lysine (I-0.5 approximate to 10 mu M), whereas the bac terial enzymes are less (50-fold) or insensitive to lysine inhibition. We f ound that plant dhdps sequences expressing lysine-sensitive DHDPS enzymes a re unable to complement a bacterial auxotroph, although a functional plant DHDPS enzyme is formed. As a consequence of this, plant dhdps cDNA clones w hich have been isolated through functional complementation using the DHDPS- deficient Escherichia coli strain encode mutated DHDPS enzymes impaired in lysine inhibition. The experiments outlined in this article emphasize that heterologous complementation can select for mutant clones when altered prot ein properties are requisite for functional rescue. In addition, the mutant s rescued by heterologous complementation revealed a new critical amino aci d substitution which renders lysine insensitivity to the plant DHDPS enzyme . An interpretation is given for the impaired inhibition mechanism of the m utant DHDPS enzyme by integrating the identified amino acid substitution in the DHDPS protein structure.