Subunit–subunit interactions are weakened in mutant forms of acetohydroxy acid synthase insensitive to valine inhibition

Abstract

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.

Interactions between the ACT Domains and Catalytic Subunits of Acetohydroxyacid Synthases (AHASs) from Different Species

“…The CSs contained three conserved negative residues (D120, E124, and D126 for Ec CSI; Figures E and S9), which might contribute to the negatively charged central hole. Consistent with our suggestion, residue E124 was identified to be crucial for subunit interaction by the yeast two‐hybrid system . Taken together, we illustrated the most probable orientation of CS and RS and predicted the structure of the AHAS complex (Figure G).…”

Adaptation of an L-Proline Adenylation Domain to Use 4-Propyl-L-Proline in the Evolution of Lincosamide Biosynthesis

“…The lmbC gene was excised via the Nde I and Xho I restriction sites from plmbC1 and inserted into a pJAKO [62] cloning vector, derived from pBluescript II KS+ (Stratagene) using the same restriction sites. The resulting plmbC2 plasmid was used for in vitro site-directed mutagenesis using the QuickChange Site-Directed Mutagenesis Kit (Stratagene) and mutagenic primers G308Vf 5’-CAACATCTAC GG T CCGACCGAGACCAACG T CTGTACGTACG-3’ and G308Vr 5’-CGTACGTACAGACGTTGGTCTCGGTCGGACCGTAGATGTTG-3’.…”

Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16

“…Furthermore, these constructs do not bind valine or respond to BCAA-inhibition Mendel et al 2003;Mendel et al 2001). It is unclear how both the N and C terminus of IlvH interact with BCAAs, although studies hypothesized that the mutant forms of AHAS has a weakened IlvH subunit interaction (Kyselková et al 2010). Additional potential mutations that might contribute to lack of valine sensitivity may exist, although not generated in this study.…”

“…An alignment of the amino acid sequences of the small subunits of three valine-sensitive AHASs of E. coli and R. eutropha, and valine-insensitive AHAS II from E. coli was conducted and revealed ten amino acid residues (N11, G14, G21, L22, F23, N29, E31, P38, E58, and Q59) that were found to be present only in valine-sensitive AHASs (Online resource 7). It was hypothesized that the N-terminus of IlvH could be responsible for binding valine (Eoyang and Silverman 1986;Kyselková et al, 2010;Leyval et al, 2003), and therefore these conserved residues were replaced individually with residues found in valineinsensitive AHAS II (N11F, G14E, G21R, L22V, F23V, N29H, E31C, and P38A). The resulting IlvH expression plasmids are listed in Table 1.…”