Chemoenzymatic Synthesis of an Unnatural Deazaflavin Cofactor That Can Fuel F₄₂₀-Dependent Enzymes

Abstract

F 420 -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 F 420 -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 reached 80% of FO conversion in 12 h. The same enzyme could produce 1 mg (2.5 μmol) of FOP in 50 mL of reaction volume, which translates to a production of 50 μmol/L. The activity toward FOP was tested for an enzyme of each of the three main structural classes of F 420 -dependent oxidoreductases. The sugar-6-phosphate dehydrogenase from Cryptosporangium arvum (FSD-Cryar), the F 420 :NADPH oxidoreductase from Thermobif ida fusca (TfuFNO), and the F 420 -dependent reductases from Mycobacterium hassiacum (FDR-Mha) all showed activity for FOP. Although the activity for FOP was lower than that for F 420 , with slightly lower k cat and higher K m values, the catalytic efficiencies were only 2.0, 12.6, and 22.4 times lower for TfuFNO, FSD-Cryar, and FDR-Mha, respectively. Thus, FOP could be a serious alternative for replacing F 420 and might boost the application of F 420 -dependent enzymes in biocatalysis.

Cofactor F420-Dependent Enzymes: An Under-Explored Resource for Asymmetric Redox Biocatalysis

“…The ability of the mycobacterial FDORs to reduce activated C=C double bonds was first identified when DDN was shown to be responsible for activating the bicyclic nitroimidazole PA-824 in M. tuberculosis. These enzymes were then shown to also reduce enoates in aflatoxins, coumarins, furanocoumarins and quinones [6,12,14,16,[34][35][36][37][38]. Recent studies have shown that these enzymes are promiscuous and can use cyclohexen-1-one, malachite green and a wide range of other activated ene compounds as substrates [35].…”

“…FGD from Rhodococcus jostii and Mycobacterium smegmatis have been studied and expressed in E. coli, both the enzymes were stable in in vitro assays [26,39,60]. Both FGDs have been expressed in engineered E. coli producing cofactor F 420 together with FDORs [38,59] FGDs have been shown to efficiently regenerate reduced cofactor F 420 both in vivo and in vitro. However, the cost of the glucose-6-phosphate and the need to separate reaction products from the accumulated FGD byproduct (6-phosphoglucono-d-lactone) may prove to be impediments for the adoption of FGD as a recycling system for cofactor F 420 in the in vitro biotransformations.…”

“…Another recent advance is the production of a synthetic analog of cofactor F 420 , called F O -5 -phosphate (F O P). F O P was derived from F O , the metabolic precursor of cofactor F 420 , which is phosphorylated using an engineered riboflavin kinase [38]. F O P has also been shown to function as an active cofactor for cofactor F 420 -dependent enzymes activities, albeit there is a penalty in the rates of these reactions [38].…”

“…F O P was derived from F O , the metabolic precursor of cofactor F 420 , which is phosphorylated using an engineered riboflavin kinase [38]. F O P has also been shown to function as an active cofactor for cofactor F 420 -dependent enzymes activities, albeit there is a penalty in the rates of these reactions [38]. Drenth and coworkers prepared F O by chemical synthesis, using a method developed by Hossain et al [77].…”

“…However, it is likely that the engineered kinase for the phosphorylation of F O could be introduced into an organism that over-produces F O allowing for the production of F O P by fermentation. This semisynthetic pathway would have the advantage that it needs only two biosynthetic steps, instead of the four steps needed for cofactor F 420 production, and demands less metabolic input from the native host metabolism (e.g., no glutamate is required) [38]. The production of F O P also opens the possibility of making deazaflavin analogs of FMN and FAD, which would be electrochemically more like F 420 than flavins, but may still bind FMN and FAD-dependent enzymes and potentially allow access to new chemistry with already well-characterized enzymes.…”