Structure of the ask-asd operon and formation of aspartokinase subunits inthe cephamycin producer 'Amycolatopsis lactamdurans'

The first two genes of the lysine pathway are closely linked forming a tran scriptional operon in the cephamycin producer 'Amycolatopsis lactamdurans'. The asd gene, encoding the enzyme aspartic semialdehyde dehydrogenase. has been cloned by complementation of Escherichia coli asd mutants. It encodes a protein of 355 aa with a deduced M-r of 37109, The ask gene encoding the aspartokinase (Ask) is located upstream of the asd gene as shown by determ ination of Ask activity conferred to E. coli transformants, asd and ask are separated by 2 nt and are transcribed in a bicistronic 2.6 kb mRNA, As occ urs in corynebacteria, the presence of a ribosome-binding site within the a sk sequence suggests that this ORF encodes two overlapping proteins, Ask al pha of 421 aa and M-r 44108. and Ask beta of 172 aa and M-r 18145. The form ation of both subunits of Ask from a single gene (ask) was confirmed by usi ng antibodies against the C-terminal end of Ask which is identical in both subunits. Ask activity of 'A. lactamdurans' is regulated by the concerted a ction of lysine plus threonine and this inhibition is abolished in E. coli transformants containing Ser(301) to Tyr, or Gly(345) to Asp mutations of t he 'A. lactamdurans' ask gene.