Project description: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. strain BQ1 degrades NMP. By studying a transposon-tagged mutant unable to degrade NMP, we identified a six-gene cluster (nmpABCDEF) that is transcribed as a polycistronic mRNA and encodes enzymes involved in NMP biodegradation. nmpA and the transposon-affected gene nmpB encode an N-methylhydantoin amidohydrolase that transforms NMP to ?-N-methylaminobutyric acid; this is metabolized by an amino acid oxidase (NMPC), either by demethylation to produce ?-aminobutyric acid (GABA) or by deamination to produce succinate semialdehyde (SSA). If GABA is produced, the activity of a GABA aminotransferase (GABA-AT), not encoded in the nmp gene cluster, is needed to generate SSA. SSA is transformed by a succinate semialdehyde dehydrogenase (SSDH) (NMPF) to succinate, which enters the Krebs cycle. The abilities to consume NMP and to utilize it for growth were complemented in the transposon-tagged mutant by use of the nmpABCD genes. Similarly, Escherichia coli MG1655, which has two SSDHs but is unable to grow in NMP, acquired these abilities after functional complementation with these genes. In wild-type (wt) BQ1 cells growing in NMP, GABA was not detected, but SSA was present at double the amount found in cells growing in Luria-Bertani medium (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 ?-N-methylaminobutyric acid is deaminated to SSA instead of being demethylated to GABA.IMPORTANCEN-Methylpyrrolidone is a cyclic amide reported to be biodegradable. However, the metabolic pathway and enzymatic activities for degrading NMP are unknown. By developing molecular biology techniques for Alicycliphilus sp. strain 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 applications in bioremediation.
