FAD is a preferred substrate and an inhibitor of Escherichia coli general NAD(P)H:flavin oxidoreductase

Escherichia coli general NAD(P)H:flavin oxidoreductase (Fre) does not have a bound flavin cofactor; its flavin substrates (riboflavin, FMN, and FAD) are believed to bind to it mainly through the isoalloxazine ring. This interaction was real for riboflavin and FMN, but not for FAD, which bound to Fre much tighter than FMN or riboflavin. Computer simulations of Fre.FAD and Fre.FMN complexes showed that FAD adopted an unusual bent conformation, allowing its ribityl side chain and ADP moiety to form an additional 3.28 H-bonds on average with amino acid residues located in the loop connecting Fbeta5 and Falpha1 of the flavin-binding domain and at the proposed NAD(P)H-binding site. Experimental data supported the overlapping binding sites of FAD and NAD(P)H. AMP, a known competitive inhibitor with respect to NAD(P)H, decreased the affinity of Fre for FAD. FAD behaved as a mixed-type inhibitor with respect to NADPH. The overlapped binding offers a plausible explanation for the large K(m) values of Fre for NADH and NADPH when FAD is the electron acceptor. Although Fre reduces FMN faster than it reduces FAD, it preferentially reduces FAD when both FMN and FAD are present. Our data suggest that FAD is a preferred substrate and an inhibitor, suppressing the activities of Fre at low NADH concentrations.     

Characterization of fatty acids and proteins associated with the xanthophyll-enriched membrane fraction isolated from the thylakoid membranes of irradiance-stressed Dunaliella salina

It has been previously reported that a considerable amount of lutein and zeaxanthin could be fractionated, upon mild detergent treatment, from the thylakoid membranes of irradiance-stressed unicellular green alga, Dunaliella salina, into a yellow pellet fraction. Such membrane pellet was found to be devoid of chlorophylls and any known proteins of photosynthesis but rather contained a significant amount of unknown polypeptides. It was speculated that this xanthophyll-rich membrane pellet might originate from incomplete solubilization of the photoinhibited thylakoids by weak surfactants, due to extra rigidity imposed by the xanthophylls being directly imbedded into the lipid bilayer. In this study, we further characterized this membrane fraction by studying its associated proteins and fatty acid composition. Analysis by gas chromatography–mass spectrometry indicated that this yellow pellet membrane was enriched in saturated fatty acids, supporting the rigidity notion of the pellet. Protein identification by MALDI-TOF MS further revealed that at least 20 water-soluble proteins were found in association with this pellet. These proteins may originate from unspecific contamination of abundant polypeptides co-precipitated with the membrane upon fractionation. Possible explanations regarding the nature of this xanthophyll-rich membrane are also discussed.