(489f) A Blue Fluorescent Protein with Oxidoreductase Activity

Fluorescent proteins revolutionized the study of proteins, organelles, and metabolic pathways by making it possible to visualize events within cells. In 2001, Su et al identified a blue fluorescent protein from Vibrio vulnificus (Bfpvv) that does not adopt a beta-can fold like traditional fluorescent proteins, but belongs to the short-chain dehydrogenase/reductase family and obtains its fluorescence by binding and magnifying the intrinsic fluorescence of NADPH. Since the fluorescence level of Bfpvv is very low, its natural role is unlikely to be as a fluorescent protein. However, a protein with both fluorescence and activity would be a useful addition to the toolbox for protein biophysical studies as it would allow the separate monitoring of binding and catalytic events.

Despite the diverse nature of the SDR family, we were able to identify redox substrates on which Bfpvv shows considerable activity. The enzyme is highly active in the reduction of aldehydic substrates, particularly those with high hydrophobicity (e.g. aromatic and long chain hydrocarbon). The activity with ketones is considerably lower, as is the activity in the oxidative direction. When oxidizing substrates, the enzyme shows more than 15-fold preference for the S-enantiomer. The substrate profile of the enzyme suggests that the active site is partially impaired and this is what likely led to the development of fluorescent properties. Mutants with increased fluorescence have accumulated mutations in areas that affect the protein activity, further corroborating that fluorescence comes at the expense of activity. The unique properties of Bfpvv allow for both visual levels of fluorescence to monitor binding of NADPH and measurable levels of redox activity to monitor catalytic turnover.