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 chrysogenum by hybridization with a probe internal to the Penicillium chrysogenum lys2 gene, was able to complement an alpha-aminoadipate reductase-deficient 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 activation and reduction of alpha-aminoadipic acid to its semialdehyde, as shown by reaction of the product with p-dimethylaminobenzaldehyde. The reaction required 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 protein contained all nine motifs that are conserved in the adenylating domain of this enzyme family, a peptidyl carrier domain, and a reduction domain. In addition, a new NADP-binding motif located at the N-terminus of the reduction domain that may form a Rossmann-like betaalphabeta-fold has been identified and found to be shared by all known Lys2 proteins. The lys2 gene was mapped to chromosome I (2.2 Mb, the smallest chromosome) of A. chrysogenum C10 (the chromosome that contains the "late" cephalosporin cluster) and is transcribed as a monocistronic 4.5-kb mRNA although at relatively low levels compared with the beta-actin gene.
Institute of Biotechnology INBIOTEC, Science Park of León, Spain.
M J
MJ
Hijarrubia
J F
JF
Aparicio
J
J
Casqueiro
J F
JF
Martín
D000164:Acremonium / Q000201:enzymology* / Q000235:genetics*; D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D006801:Humans; D050647:L-Aminoadipate-Semialdehyde Dehydrogenase; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010408:Penicillium chrysogenum / Q000201:enzymology / Q000235:genetics; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D009243:NAD; D009249:NADP; D000445:Aldehyde Oxidoreductases; D050647:L-Aminoadipate-Semialdehyde Dehydrogenase