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Geny kodující acetyhydroxy syntázu u Streptomyces cinnamonensis a jejich regulační oblast:Mutace v katalytické podjednotce zjią»ující necitlivost k inhibici konečného produktu
Kyselková, Martina ; Kopecký, Jan ; Šigutová, Lucie ; Pospíšil, Stanislav ; Felsberg, Jürgen ; Spížek, Jaroslav ; Janata, Jiří
Reaction of acetohydroxy acid synthase (AHAS) is the key step in branched-chain amino acids biosynthesis. AHAS is feed-back inhibited by valin that binds its regulatory subunit. In Streptomyces cinnamonensis, production of a secondary metabolite, polyether antibiotic monensin A, is associated with valin biosynthesis via 2-oxoisovalerate. Thus, mutants with higher AHAS levels or AHAS insensitive to valin show increased monensin A production. Gene ilvB coding for the AHAS catalytic subunit of the S. cinnamonensis parental strain and four mutants resistant to valin analogues was cloned and sequenced. Nucleotide sequence of ilvB from the strain BVR-18 differed from the wild type in a single substitution C574A resulting in an amino acid change E139A. In the ACB-NLR-2 strain, a triplet AGC in the position 805-807 was deleted resulting in the deletion of Q217. A homology model of the catalytic subunit, created by a virtue of the known three-dimensional structure of the yeast AHAS catalytic subunits dimer, revealed that the mutation found in the BVR-18 was located in a loop at the subunit-subunit interface in a close proximity of the TPP binding site in the active centre. The Q217 deletion in the ACB-NLR-2 occurred in a helix distant from the catalytic site, and seemed to shorten it. AHAS assays in a crude cell extract from the S. cinnamonensis BVR-18 and ACB-NLR-2 strains showed not only the insensitivity to valin but even higher activities of both enzymes in the presence of 10 mM valine. Catalytic and regulatory subunits from the parental and mutant strains were separately expressed in E. coli and combined in vitro to reconstitute the holoenzyme. Properties of such in vitro prepared enzymes were tested. The ACBR-2 and NLR-3 strains with increased AHAS levels have no mutation in the AHAS catalytic subunit. The ilvB regulatory region of those two strains and of the parental strain was cloned and sequenced using the method of chromosome walking. A putative leader peptide and a transcription termination sequence were identified within this region indicating that ilvB expression is controlled by attenuation. However, neither the ACBR-2, nor the NLR-3 have any mutation in the ilvB regulatory region suggesting that some other regulatory mechanism may participate in ilvB expression.
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