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Chemoenzymatic Synthesis of an Unnatural Deazaflavin Cofactor That Can Fuel F-420-Dependent Enzymes
- Source :
- ACS Catalysis, 9(7), 6435-6443. AMER CHEMICAL SOC
- Publication Year :
- 2019
- Publisher :
- AMER CHEMICAL SOC, 2019.
-
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 mu mol 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 mu mol) of FOP in 50 mL of reaction volume, which translates to a production of 50 mu mol/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 Thermobifida 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.
- Subjects :
- NADP REDUCTASE
biocatalysis
010402 general chemistry
Riboflavin kinase
COENZYME F-420
TUBERCULOSIS
01 natural sciences
Redox
GLUCOSE-6-PHOSPHATE-DEHYDROGENASE
Catalysis
Cofactor
riboflavin kinase
Oxidoreductase
SECONDARY ALCOHOL-DEHYDROGENASE
F-420 PROTECTS MYCOBACTERIA
DRUG CANDIDATE
OXIDOREDUCTASE
F-420
chemistry.chemical_classification
PURIFICATION
biology
010405 organic chemistry
Drug candidate
food and beverages
General Chemistry
Combinatorial chemistry
0104 chemical sciences
CATALYZE
Enzyme
chemistry
deazaflavin
Biocatalysis
reductase
biology.protein
Subjects
Details
- Language :
- English
- ISSN :
- 21555435
- Volume :
- 9
- Issue :
- 7
- Database :
- OpenAIRE
- Journal :
- ACS Catalysis
- Accession number :
- edsair.doi.dedup.....845e53856221bacc9b4de25dcf5503a1