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Fresh Surprises from the old Cofactor NAD(P)H
Author(s) -
Rosenthal Raoul,
Vögeli Bastian,
Stoffel Gabriele,
Erb Tobias
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.573.9
Subject(s) - cofactor , nad+ kinase , chemistry , stereochemistry , enzyme , protonation , redox , dehydrogenase , substrate (aquarium) , enzyme catalysis , hydride , active site , catalytic cycle , biochemistry , organic chemistry , biology , ion , ecology , hydrogen
About 16% of all characterized enzymes use the pyridine nucleotides NADH or NADPH as hydride donors or acceptors during catalysis. The textbook mechanism for these cofactors is a direct hydride transfer between NAD(P)H and the corresponding substrate. This long‐standing mechanistic hypothesis was recently challenged by the surprising discovery of an intermediate during catalysis of two NADPH‐dependent enzymes of the middle chain dehydrogenase reductase superfamily (Rosenthal et al. Nature Chem. Biol. 2014). Stopped‐flow spectroscopy, NMR and high resolution MS revealed that the intermediate in these enyzmes is a covalent ene‐adduct between the substrate crotonyl‐CoA and the NADPH‐cofactor. These observations provide ‐for the first time‐ evidence that NAD(P)H‐dependent enzymes can react through an ene‐mechanism instead of the commonly assumed direct hydride transfer.Subsequent experiments allowed us to isolate the catalytic intermediate and use it as a novel tool to dissect the individual steps of catalysis. With this strategy we could directly study the protonation half‐reaction of fatty acid reductases and identify the long‐sought enigmatic proton donor in these enzymes. Having understood the structure function relationships in the active site we could engineer the enzyme to have an altered stereochemistry (Rosenthal et al. submitted). These results have implications for our fundamental understanding of Nature's most abundant redox cofactor and for enzyme engineering efforts in controlling stereo chemistry of reduction reactions.