BACKGROUND: This study investigates the mechanisms of resistance to acetolactate synthase-inhibiting herbicides in populations of Apera spica-venti (L.) P.B. from the Czech Republic. RESULTS: The proportion of resistance due to mutant acetolactate synthase (ALS) alleles was estimated by genotyping individuals from each of three populations for the eight ALS mutations known to confer resistance. Four resistance-conferring ALS mutations were identified: Pro-197-Ala, Pro-197-Thr, Trp-574-Leu and previously unreported Trp-574-Met substitution. Two populations (R1, R3) have amino acid substitution at positions Pro-197 and Trp-574. Individuals from the R3 population had two different resistance alleles. In the R2 population, only the resistant Trp-574-Met substitution was detected. Ten other single point mutations were identified, but these were not related to resistance. The cytochrome malathion decreased chlorsulfuron resistance in the resistant populations that were examined. Although malathion increased mortality, the GR50 values were too high to conclude that non-target-based mechanism was the main one for the resistance in Apera spica-venti populations tested in this study. CONCLUSIONS: Individuals of Apera spica-venti populations tested in this study possess the target-site ALS resistance mutation and an additional so far unknown resistance mechanism(s).
- MeSH
- acetolaktátsynthasa biosyntéza MeSH
- DNA rostlinná MeSH
- fyziologická adaptace genetika MeSH
- inhibitory enzymů toxicita MeSH
- lipnicovité genetika MeSH
- malathion toxicita MeSH
- molekulární sekvence - údaje MeSH
- rezistence k herbicidům genetika MeSH
- sekvence nukleotidů MeSH
- sulfonamidy toxicita MeSH
- triaziny toxicita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
In acetohydroxy acid synthase from Streptomyces cinnamonensis mutants affected in valine regulation, the impact of mutations on interactions between the catalytic and the regulatory subunits was examined using yeast two-hybrid system. Mutations in the catalytic and the regulatory subunits were projected into homology models of the respective proteins. Two changes in the catalytic subunit, E139A (alpha domain) and DeltaQ217 (beta domain), both located on the surface of the catalytic subunit dimer, lowered the interaction with the regulatory subunit. Three consecutive changes in the N-terminal part of the regulatory subunit were examined. Changes G16D and V17D in a loop and adjacent alpha-helix of ACT domain affected the interaction considerably, indicating that this region might be in contact with the catalytic subunit during allosteric regulation. In contrast, the adjacent mutation L18F did not influence the interaction at all. Thus, L18 might participate in valine binding or conformational change transfer within the regulatory subunits. Shortening of the regulatory subunit to 107 residues reduced the interaction essentially, suggesting that the C-terminal part of the regulatory subunit is also important for the catalytic subunit binding.
Acetohydroxy-acid synthases (AHAS) of two mutant strains Streptomyces cinnamonensis ACB-NLR-2 and BVR-18 were chosen for this study for their apparent activation by valine, which regularly acts as an allosteric inhibitor. Sequencing the ilvB genes coding for the AHAS catalytic subunit revealed two distant changes in the mutants, DeltaQ217 and E139A, respectively. Homology modeling was used to propose the structural changes caused by those mutations. In the mutant strain ACB-NLR-2 (resistant to 2-amino-3-chlorobutyrate and norleucine), deletion of Q217 affected a helix in ss-domain, distant from the active center. As no mutation was found in the regulatory subunit of this strain, DeltaQ217 in IlvB was supposed to be responsible for the observed valine activation, probably via changed properties on the proposed regulatory-catalytic subunit interface. In mutant strain BVR-18 (resistant to 2-oxobutyrate), substitution E139A occurred in a conservative loop near the active center. In vitro AHAS activity assay with the enzyme reconstituted from the wild-type regulatory and BVR-18 catalytic subunits proved that the substitution in the catalytic subunit led to the apparent activation of AHAS by valine. We suggest that the conservative loop participated in a conformational change transfer to the active center during the allosteric regulation.
- MeSH
- acetolaktátsynthasa genetika chemie metabolismus MeSH
- aktivace enzymů MeSH
- alosterická regulace imunologie MeSH
- bakteriální proteiny genetika chemie metabolismus MeSH
- bodová mutace MeSH
- katalytická doména imunologie MeSH
- konformace proteinů MeSH
- missense mutace MeSH
- molekulární modely MeSH
- rekombinantní fúzní proteiny chemie metabolismus MeSH
- sekvenční homologie aminokyselin MeSH
- Streptomyces enzymologie genetika MeSH
- substituce aminokyselin MeSH
- valin metabolismus MeSH
- vztahy mezi strukturou a aktivitou MeSH
- MeSH
- acetolaktátsynthasa antagonisté a inhibitory genetika metabolismus MeSH
- bakteriální proteiny genetika metabolismus MeSH
- Corynebacterium enzymologie genetika růst a vývoj MeSH
- regulace genové exprese u bakterií MeSH
- sekvence aminokyselin MeSH
- techniky in vitro MeSH
- valin biosyntéza MeSH
- větvené aminokyseliny farmakologie MeSH
