A mechanism for valine-resistant growth of Escherichia coli K-12 supported by the valine-sensitive acetohydroxy acid synthase IV activity from ilvJ662

Add time:09/04/2019    Source:sciencedirect.com

Acetohydroxy acid synthase (EC 4.1.3.18; AHAS) isozymes I and III are expressed in Escherichia coli strain K-12 and, when inhibited by L-valine, cannot support cell growth. AHAS IV, expressed from mutation at ilvJ662, exhibits valine-sensitivity similar to that of AHAS III, yet AHAS IV does support cell growth in valine minimal medium. Rate equations were derived for AHAS III and AHAS IV reactions in crude extracts and for partially purified AHAS IV. Values of kinetic constants in these equations were determined in order to model a probable reaction mechanism. Computer modeling of initial velocity reactions at physiological substrate concentrations simulated consequences of valine-inhibition and revealed that AHAS IV synthesized AHB at a maximal rate over four times faster than AHAS III under these conditions. The simulations predicted that cells depending upon AHAS III for growth in valine minimal medium would accumulate higher levels of 2-ketobutyrate than cells using AHAS IV. Experiments on growth inhibition by valine revealed more than a five-fold difference in 2-ketobutyrate accumulation, thus confirming these predictions. These data support the hypothesis that valine inhibition of growth is a consequence of 2-ketobutyrate accumulation to toxic levels. We propose that the valine-inhibited AHAS IV activity prevents growth inhibition by keeping 2-ketobutyrate accumulation to a lower level than that resulting from AHAS III activity.

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