| Products/Services Used | Details | Operation |
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| Gene Synthesis | The C. ammoniagenes riboflavin kinase gene (CaRFK) was ordered at GenScript (Piscataway, NJ, U.S.A.), codon optimized for E. coli. It was composed of the C-terminal kinase domain of the FAD synthetase gene, ribF(NCBI#: D37967.1), previously described by Iamurri et al.(49) The nucleotide sequence and protein sequence are shown in Figures S1 and S2, respectively. The gene was cloned into a pBAD/Myc-His vector (Invitrogen, Thermo-Fisher) using restriction sites NdeI and HindIII, following standard cloning procedures.(50) Site-directed mutagenesis was performed on the riboflavin kinase gene with the use of mutagenic primers, degenerate at a chosen codon, using the QuikChange mutagenesis kit (Stratagene), following the procedure of the manufacturer. Primers were designed with the Agilent QuikChange primer design tool (http://www.genomics.agilent.com/primerDesignProgram.jsp). The used primers are listed in Table S1. Sequencing was performed at GATC (Constanz, Germany). The plasmids were transformed into calcium chloride chemically competent E. coli NEB 10-beta (New England Biolabs Ipswich, MA, U.S.A.) for amplification and protein expression, using standard protocols.(50) | Get A Quote |
F420-dependent enzymes are found in many microorganisms and can catalyze a wide range of redox reactions, including those with some substrates that are otherwise recalcitrant to enzyme-mediated reductions. Unfortunately, the scarceness of the cofactor prevents application of these enzymes in biocatalysis. The best F420-producing organism, Mycobacterium smegmatis, only produces 1.4 μmol per liter of culture. Therefore, we synthesized the unnatural cofactor FO-5′-phosphate, coined FOP. The FO core-structure was chemically synthesized, and an engineered riboflavin kinase from Corynebacterium ammoniagenes(CaRFK) was then used to phosphorylate the 5′-hydroxyl group. The triple F21H/F85H/A66I CaRFK mutant reac... More
