Deregulation of acetohydroxy-acid synthase: Loss of allosteric inhibition conferred by mutations in the catalytic subunit
Language English Country United States Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- Acetolactate Synthase chemistry genetics metabolism MeSH
- Enzyme Activation MeSH
- Allosteric Regulation genetics MeSH
- Bacterial Proteins chemistry genetics metabolism MeSH
- Point Mutation MeSH
- Catalytic Domain genetics MeSH
- Protein Conformation MeSH
- Mutation, Missense MeSH
- Models, Molecular MeSH
- Recombinant Fusion Proteins chemistry metabolism MeSH
- Sequence Homology, Amino Acid MeSH
- Streptomyces enzymology genetics MeSH
- Amino Acid Substitution MeSH
- Valine metabolism MeSH
- Structure-Activity Relationship MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Acetolactate Synthase MeSH
- Bacterial Proteins MeSH
- Recombinant Fusion Proteins MeSH
- Valine MeSH
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.
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