Characterization of the lys2 gene of Acremonium chrysogenum encoding a functional alpha-aminoadipate activating and reducing enzyme

A 5.2-kb NotI DNA fragment isolated from a genomic library of Acremonium ch rysogenum by hybridization with a probe internal to the Penicillium chrysog enum lys2 gene, was able to complement an alpha -aminoadipate reductase-def icient mutant of P. chrysogenum (lysine auxotroph L(-)G(-)). Enzyme assays showed that the alpha -aminoadipate reductase activity was restored in all the transformants tested. The lys2-encoded enzyme catalyzed both the activa tion and reduction of a-aminoadipic acid to its semialdehyde, as shown by r eaction of the product with p-dimethylaminobenzaldehyde. The reaction requi red NADPH, and was not observed in the presence of NADH. Sequence analysis revealed that the gene encodes a protein with relatively high similarity to members of the superfamily of acyladenylate-forming enzymes. The Lys2 prot ein contained all nine motifs that are conserved in the adenylating domain of this enzyme family, a peptidyl carrier domain, and a reduction domain. I n addition, a new NADP-binding motif located at the N-terminus of the reduc tion domain that may form a Rossmann-like beta alpha beta -fold has been id entified and found to be shared by all known Lys2 proteins. The lys2 gene w as mapped to chromosome I (2.2 Mb, the smallest chromosome) of A. chrysogen um C10 (the chromosome that contains the "late" cephalosporin cluster) and is transcribed as a monocistronic 4.5-kb mRNA although at relatively low le vels compared with the beta -actin gene.