Objective: To study dehydrogenation by short ethoxy chain nonylphenols dehydrogenase (sNPEO-DH) from Ensifer sp. AS08.
Methods: We screened four amino acid residues of sNPEO-DH that are adjacent to the isoalloxazine ring of the coenzyme flavin adenine dinucleotide (FAD) by using multiple sequence alignment and homology modeling. Mutations were introduced by site-directed mutagenesis. The recombinant proteins were expressed, purified and assayed.
Results: The relative activities of mutants N90A and N509A against the hydrophilic substrate PEG1000 decreased to 51% and 89%, respectively, and against the hydrophobic substrate NPEO(av10) decreased to 26% and 40%, respectively; indicating that N90 and N509 might be related to substrate binding. The relative activity of mutant H465A and N507A lost 90% and 100%, respectively; "stop-flow" experiments revealed that the processes of proton transfer from substrate to FAD and from FAD to enzyme were blocked in mutant N507A and H465A, respectively.
Conclusion: Amino acid residues N507 and H465 located at the activity center of sNPEO-DH and play roles as catalytic sites for the oxidative dehydrogenation of the substrates and FADH2, respectively.