Structure of the ask-asd operon and formation of aspartokinase subunits in the cephamycin producer 'Amycolatopsis lactamdurans'.
The first two genes of the lysine pathway are closely linked forming a transcriptional 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 determination 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 occurs in corynebacteria, the presence of a ribosome-binding site within the ask sequence suggests that this ORF encodes two overlapping proteins, Askalpha of 421 aa and M(r) 44108, and Askbeta of 172 aa and M(r) 18145. The formation of both subunits of Ask from a single gene (ask) was confirmed by using antibodies against the C-terminal end of Ask which is identical in both subunits. Ask activity of 'A. lactamdurans' is regulated by the concerted action 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 the 'A. lactamdurans' ask gene.
Hernándo-Rico
Victor
V
Area de Microbiologı́a, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain1.
Martı N
Juan F
JF
Area de Microbiologı́a, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain1.
Santamarta
Irene
I
Area de Microbiologı́a, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain1.
Liras
Paloma
P
Area de Microbiologı́a, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain1.
D000595:Amino Acid Sequence; D001223:Aspartate-Semialdehyde Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D002513:Cephamycins / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005796:Genes; D005816:Genetic Complementation Test; D015151:Immunoblotting; D008239:Lysine / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D009615:Nocardia / Q000235:genetics* / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D016366:Open Reading Frames; D009876:Operon; D021122:Protein Subunits; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000378:metabolism
D002513:Cephamycins; D021122:Protein Subunits; D013912:Threonine; D001223:Aspartate-Semialdehyde Dehydrogenase; D008239:Lysine