ABSTRACT
The 2-naphthoyl-coenzyme A (NCoA) reductase (NCR) is so far the only characterized enzyme involved in the anaerobic degradation of the environmentally relevant polycyclic aromatic hydrocarbons. The old yellow enzyme (OYE) family member apparently reduced the nonactivated naphthyl ring to 5,6,7,8-tetrahydro-2-napthoyl-CoA (THNCoA). In this work, the candidate genes of three NCRs from the sulphate-reducing, naphthalene-degrading N47 and NaphS2 cultures were expressed in Escherichia coli. The isolated products contained flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) a [4Fe-4S] cluster and catalyzed only the two-electron reduction of NCoA to 5,6-dihydro-2-naphthoyl-CoA (5,6-DHNCoA) at a very negative E°' = -493 mV. All NCRs exhibited high NCoA-forming DHNCoA oxidase activities that are proposed to be involved in oxygen-detoxification during naphthalene degradation. Extracts of N47 and NaphS2 catalyzed the reduction of 5,6-DHNCoA to THNCoA. Genes putatively coding for 5,6-DHNCR from N47 and NaphS2 were heterologously expressed in E. coli. The enriched enzyme products specifically catalyzed the reduction of 5,6-DHNCoA to THNCoA at E°' = -375 mV. With the three NCRs and two 5,6-DHNCRs, five OYEs have been characterized that are involved in the reduction of the nonsubstituted naphthyl-ring system; these unprecedented enzymatic reactions expand our knowledge of the functional diversity of OYE.
Subject(s)
Deltaproteobacteria/metabolism , Escherichia coli/genetics , Hydrocarbons, Aromatic/metabolism , NADPH Dehydrogenase/metabolism , Naphthalenes/metabolism , Anaerobiosis , Coenzyme A/metabolism , Escherichia coli/metabolism , Flavin Mononucleotide/metabolism , Flavin-Adenine Dinucleotide/metabolism , Gene Expression , Oxidation-Reduction , Oxidoreductases/metabolism , PhylogenyABSTRACT
The ability to control mammalian genes in a synergistic mode using synthetic transcription factors is highly desirable in fields of tissue engineering, stem cell reprogramming and fundamental research. In this study, we developed a standardized toolkit utilizing an engineered CRISPR/Cas9 system that enables customizable gene regulation in mammalian cells. The RNA-guided dCas9 protein was implemented as a programmable transcriptional activator or repressor device, including targeting of endogenous loci. For facile assembly of single or multiple CRISPR RNAs, our toolkit comprises a modular RNAimer plasmid, which encodes the required noncoding RNA components.