Characterization of the lys3 Gene and a Mutant allele from Penicillium chrysogenum encoding a Bi-Functional Protein with Homoaconitase Activity and Regulatory Functions
Teves, F.; Casqueiro, J.; Naranjo, L.; Ullán, R.V.; Bañuelos, O. and Martín, J.F.
Area
de Microbiología, Facultad de Biología, Universidad de León,
24071 León
Instituto de biotecnología (INBIOTEC), Avda. del Real Nº1,
24006 León.
The lysine biosynthetic pathway has to supply large amounts of a-aminoadipic
acid for penicillin biosynthesis in Penicillium chrysogenum. In this
article, we have characterized the P. chrysogenum L2 mutant, a lysine
auxotroph that was believed to be altered in expression of some of the lysine
biosynthesis genes. The L2 mutant was found to be deficient in homoaconitase
activity. We have cloned a gene (named lys3) that complements the L2
mutation by transformation with a P. chrysogenum genomic library, constructed
in an autonomous replicating plasmid. The lys3 encoded protein showed
high identity to homoaconitases from Aspergillus nidulans, Aspergillus
fumigatus, Saccharomyces cerevisiae and Schizosaccharomyces
pombe. In addition, we cloned the mutant lys3 allele from the L2
strain that showed significantly increased transcript levels of lys1
and lys2 genes, encoding respectively homocitrate synthase and a-aminoadipate
reductase. Analysis of the mutation present in the lys3 allele from
P. chrysogenum L2 revealed a G1532 to A1532 point mutation resulting
in a Gly496 to Asp496 substitution. This mutation is located in a highly conserved
region adjacent to two of the three cysteine residues that act as ligands
to bind the iron sulfur cluster required for homoaconitase activity. The inability
to bind the iron-sulfur cluster is known to affect the regulatory properties
of homoaconitase-like iron regulatory proteins (IRPs). The Lys3 polypeptide
seems to be a bi-functional protein with characteristics that resemble those
of human IRP-1. The apo-homoaconitase (unable to bind the iron-sulfur cluster)
may work as a regulatory protein controlling expression of the other lysine
biosynthesis genes.
