Isolation and characterization of D serine deaminase constitutive mutants by utilization of D serine as sole carbon or nitrogen source

Abstract

D Serine deaminase (Dsdase) functions primarily as a detoxifying enzyme in E. coli K 12 as the inducer, D serine, is toxic at concentrations greater than 25 μg/ml. Dsdase converts DS to pyruvate and NH3 (ammonia), allowing DS to serve as the sole source of carbon and nitrogen. Mutants constitutive for Dsdase synthesis were isolated by utilizing D serine as sole nitrogen or carbon source in the chemostat. This method generated only regulatory constitutive (dsdC) mutants. The altered dsdC gene product in these strains is apparently able to bind D serine more efficiently than the wild type dsdC+ gene product, a selective advantage. Constitutive synthesis of Dsdase in all of these dsdC mutants is extremely sensitive to catabolite repression, and catabolite repression is reversed by the addition of D serine. Of the 15 mutants generated by this method, none are suppressible by supD, supE, or supF. Mutations to a low level of constitutivity (maximal specific activity of 9) occur much more frequently than mutations to a high level (maximal specific activity of 79). High level constitutive synthesis of Dsdase results from the synthesis of an altered dsdC gene product, not from loss of ability to form the dsdC product. Dsdase synthesis is not regulated by the nitrogen supply in the medium, as nitrogen starvation does not result in the derepression of Dsdase synthesis.