Lilianha Domínguez-Malfavón, Luis Lozano, M Javier Cruz-Gómez, Claudia Julieta Solís-González, Herminia Loza-Tavera, Martín Vargas-Suárez, Miguel Ángel Cevallos, Itzel Gaytán
The molecular mechanisms underlying the biodegradation of N-Methylpyrrolidone (NMP), a widely used industrial solvent that produces skin irritation in humans, and is teratogenic in rats, are unknown. Alicycliphilus sp. BQ1 degrades NMP. By studying a transposon-tagged mutant unable to degrade NMP, we identified a six-gene cluster (nmpABCDEF), transcribed as a polycistronic mRNA, encoding enzymes involved in NMP biodegradation. nmpA and the transposon-affected gene nmpB encode a N-methylhydantoin amidohydrolase that would transform NMP to γ-N-methylaminobutyric acid; this would be metabolized by an aminoacid oxidase (NMPC), either by demethylation to produce γ-aminobutyric acid (GABA), or by deamination to produce succinate semialdehyde (SSA). Whether GABA was produced, the activity of a GABA aminotransferase (GABA-AT), not encoded in the nmp gene cluster, would be needed to generate SSA. SSA would be transformed by succinate semialdehyde dehydrogenase (SSDH) (NMPF) to succinate, which enters the Krebs cycle. The ability to consume NMP and to utilize it for growth was complemented in the mutant with nmpABCD genes. Similarly, Escherichia coli MG1655, which has two SSDHs but is unable to grow in NMP, acquired this ability after functional complementation with these genes. In BQ1 wt cells growing in NMP, GABA was not detected, but SSA was twice the amount than in cells growing in LB, suggesting that GABA is not an intermediate in this pathway. Moreover, E. coli GABA-AT deletion mutants complemented with nmpABCD genes retained the ability to grow in NMP, supporting the possibility that γ-N-methylaminobutyric acid was deaminated to SSA instead of being demethylated to GABA.Importance N-Methylpyrrolidone is a cyclic amide reported as biodegradable. However, the metabolic pathway and the enzymatic activities for degrading NMP are unknown. By developing molecular biology techniques for Alicycliphilus sp. BQ1, an environmental bacterium able to grow in NMP, we identified a six-gene cluster encoding enzymatic activities involved in NMP degradation. These findings set the basis for the study of new enzymatic activities, and for the development of biotechnological processes with potential application in bioremediation.
