Analyses of variants of the Ser/Thr dehydratase IlvA provide insight into 2-aminoacrylate metabolism in .

RidA is a conserved and broadly distributed protein that has enamine deaminase activity. In a variety of organisms tested thus far, lack of RidA results in the accumulation of the reactive metabolite 2-aminoacrylate (2AA), an obligate intermediate in the catalytic mechanism of several pyridoxal 5'-phosphate (PLP)-dependent enzymes. This study reports the characterization of variants of the biosynthetic serine/threonine dehydratase (EC 4.3.1.19; IlvA), which is a significant generator of 2AA in the bacteria , , and and the yeast Two previously identified mutations, and suppressed the phenotypic growth consequences of 2AA accumulation in Characterization of the respective protein variants suggested that they affect 2AA metabolism by two different catalytic mechanisms, both leading to an overall reduction in serine dehydratase activity. To emphasize the physiological relevance of the enzyme characterization, we sought to explain phenotypes using these data. A simple mathematical model describing the impact these catalytic deficiencies had on 2AA production was generally supported by our data. However, caveats arose when kinetic parameters, determined , were used to predict formation of the isoleucine precursor 2-ketobutyrate and model (growth) behaviors. Altogether, our data support the need for a holistic approach, including and analyses, to generate data used in understanding and modeling metabolism.