Genes coding for acetyhydroxy acid synthase in Streptomyces cinnamonensis and their regulatory region:Mutations in the catalytic subunit conferring insesitivity to end-product inhibition

0108134 - MBU-M 20040338 RIV CZ eng C - Conference Paper (international conference)
Kyselková, Martina - Kopecký, Jan - Šigutová, Lucie - Pospíšil, Stanislav - Felsberg, Jürgen - Spížek, Jaroslav - Janata, Jiří
Genes coding for acetyhydroxy acid synthase in Streptomyces cinnamonensis and their regulatory region:Mutations in the catalytic subunit conferring insesitivity to end-product inhibition.
[Geny kodující acetyhydroxy syntázu u Streptomyces cinnamonensis a jejich regulační oblast:Mutace v katalytické podjednotce zjią»ující necitlivost k inhibici konečného produktu.]
Kongres Československé společnosti mikrobiologické /23./. 2004, s. 210.
[Kongres československé společnosti mikrobiologické /23./. Brno (CZ), 06.09.2004-09.09.2004]
Institutional research plan: CEZ:AV0Z5020903
Keywords : streptomyces sinnamonensis
Subject RIV: EE - Microbiology, Virology

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.

Geny ilvB kódující katalytickou podjednotku AHAS u rodičovského kmene S. cinnamonensis a čtyř mutantů s deregulovanou biosyntetickou drahou valinu byly sekvenovány. Mutace byly nalezeny u dvou kmenů, jejichž AHAS byla v hrubém buněčném extraktu zdánlivě aktivována valinem. U kmene BVR-18 byla nalezena mutace C574A vedoucí v proteinu k substituci E139A. U kmene ACB-NLR-2 došlo k deleci tripletu AGC v pozici 805-807, což v proteinu vedlo k deleci Q217. Podle homologního modelu katalytické podjednotky, vytvořeného na základě krystalové struktury dimeru katalytických podjednotek AHAS u Saccharomyces cerevisiae, se substituce E139A vyskytla ve smyčce na rozhraní podjednotek poblíž TPP vazebného místa, zatímco ?Q217 v helixu odlehlém od aktivního místa. Jednotlivé podjednotky byly exprimovány v E. coli za účelem rekonstituce enzymů in vitro, jejichž aktivita v přítomnosti a nepřítomnosti 10 mM valinu byla měřena. Podařilo se prokázat, že substituce E139A je zodpovědná za zdánlivou aktivaci AHAS valinem pozorovanou u kmene BVR-18. U kmenů ACBR-2 a NLR-3 se zvýšenou hladinou aktivity AHAS nebyla zjištěna žádná mutace v katalytické podjednotce. Regulační oblast před genem ilvB těchto dvou kmenů a kmene rodičovského byla sekvenována. V rámci této oblasti byly nalezeny některé prvky atenuátoru, ale sekvence se u kmenů ACBR-2 a NLR-3 shodovala se sekvencí rodičovského kmene. Zdá se tedy, že regulace exprese AHAS je zajištěna ještě jiným mechanismem, než je atenuace.
Permanent Link: http://hdl.handle.net/11104/0015273