Role of small subunit (IlvN polypeptide) of acetohydroxyacid synthase I from Escherichia coli K-12 in sensitivity of the enzyme to valine inhibition.
Most of the coding sequence for the IlvN polypeptide subunit of acetohydroxyacid synthase I was deleted from the ilvB+ ilvN+ plasmid pTCN12 by in vitro methods. Several ilvB+ delta ilvN derivatives of pTCN12 were identified among transformants of a strain otherwise lacking any acetohydroxyacid synthase. Deletion derivatives produced an enzymatically active IlvB polypeptide, as shown by the Ilv+ phenotype of transformed cells and by immunologic and enzymatic assays. However, whereas the growth of pTCN12 transformants was sensitive to valine inhibition, growth of the ilvB+ delta ilvN transformants was relatively resistant. Moreover, in vitro analyses confirmed that both acetolactate and acetohydroxybutyrate synthesis in extracts of the ilvB+ delta ilvN transformants was resistant to valine inhibition, in comparison with that in extracts of pTCN12 transformants or with that catalyzed by purified acetohydroxyacid synthase I. The IlvN polypeptide had a minimal effect, if any, on IlvB polypeptide accumulation as measured by immunoprecipitation, but its absence resulted in a greater than 10-fold reduction in enzyme specific activity.
D000095:Acetolactate Synthase / Q000037:antagonists & inhibitors*; D000595:Amino Acid Sequence; D001483:Base Sequence; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000201:enzymology*; D006885:Hydroxybutyrates / Q000096:biosynthesis; D007773:Lactates / Q000096:biosynthesis; D046911:Macromolecular Substances; D007652:Oxo-Acid-Lyases / Q000037:antagonists & inhibitors*; D010957:Plasmids; D014633:Valine / Q000494:pharmacology*
D006885:Hydroxybutyrates; D007773:Lactates; D046911:Macromolecular Substances; C006359:alpha-acetolactate; D000095:Acetolactate Synthase; D004262:DNA Restriction Enzymes; D007652:Oxo-Acid-Lyases; D014633:Valine