Solís-González Claudia Julieta, Domínguez-Malfavón Lilianha, Vargas-Suárez Martín, Gaytán Itzel, Cevallos Miguel Ángel, Lozano Luis, Cruz-Gómez M Javier, Loza-Tavera Herminia
Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, Mexico City, México.
Programa de Genómica Evolutiva, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México.
Appl Environ Microbiol. 2017 Dec 15;84(1). doi: 10.1128/AEM.02136-17. Print 2018 Jan 1.
The molecular mechanisms underlying the biodegradation of -methylpyrrolidone (NMP), a widely used industrial solvent that produces skin irritation in humans and is teratogenic in rats, are unknown. sp. strain BQ1 degrades NMP. By studying a transposon-tagged mutant unable to degrade NMP, we identified a six-gene cluster () that is transcribed as a polycistronic mRNA and encodes enzymes involved in NMP biodegradation. and the transposon-affected gene encode an -methylhydantoin amidohydrolase that transforms NMP to γ--methylaminobutyric acid; this is metabolized by an amino acid oxidase (NMPC), either by demethylation to produce γ-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 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 genes. Similarly, 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, GABA-AT deletion mutants complemented with genes retained the ability to grow in NMP, supporting the possibility that γ--methylaminobutyric acid is deaminated to SSA instead of being demethylated to GABA.-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 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.
N-甲基吡咯烷酮（NMP）是一种广泛使用的工业溶剂，会对人类皮肤产生刺激，对大鼠具有致畸性，其生物降解的分子机制尚不清楚。sp.菌株BQ1可降解NMP。通过研究一个不能降解NMP的转座子标签突变体，我们鉴定出一个六基因簇（），该基因簇转录为多顺反子mRNA，并编码参与NMP生物降解的酶。和受转座子影响的基因编码一种N-甲基乙内酰脲酰胺水解酶，该酶将NMP转化为γ-N-甲基氨基丁酸；这由一种氨基酸氧化酶（NMPC）代谢，要么通过去甲基化产生γ-氨基丁酸（GABA），要么通过脱氨基产生琥珀酸半醛（SSA）。如果产生GABA，则需要基因簇中未编码的GABA转氨酶（GABA-AT）的活性来生成SSA。SSA由琥珀酸半醛脱氢酶（SSDH）（NMPF）转化为琥珀酸，后者进入三羧酸循环。在转座子标签突变体中，通过使用基因补充了消耗NMP并将其用于生长的能力。同样，MG1655有两个SSDH，但不能在NMP中生长，在与这些基因进行功能互补后获得了这些能力。在NMP中生长的野生型（wt）BQ1细胞中，未检测到GABA，但SSA的含量是在Luria-Bertani培养基（LB）中生长的细胞中发现的两倍，这表明GABA不是该途径的中间产物。此外，用基因互补的GABA-AT缺失突变体保留了在NMP中生长的能力，支持γ-N-甲基氨基丁酸脱氨基生成SSA而不是去甲基化生成GABA的可能性。N-甲基吡咯烷酮是一种据报道可生物降解的环状酰胺。然而，降解NMP的代谢途径和酶活性尚不清楚。通过为能够在NMP中生长的环境细菌sp.菌株BQ1开发分子生物学技术，我们鉴定出一个编码参与NMP降解的酶活性的六基因簇。这些发现为研究新的酶活性以及开发在生物修复中具有潜在应用的生物技术过程奠定了基础。
