Swapping FAD binding motifs between plastidic and bacterial ferredoxin-NADP(H) reductases.

Plant-type ferredoxin-NADP(H) reductases (FNRs) are grouped in two classes, plastidic with an extended FAD conformation and high catalytic rates and bacterial with a folded flavin nucleotide and low turnover rates. The 112-123 ß-hairpin from a plastidic FNR and the carboxy-terminal tryptophan of a bacterial FNR, suggested to be responsible for the FAD differential conformation, were mutually exchanged. The plastidic FNR lacking the ß-hairpin was unable to fold properly. An extra tryptophan at the carboxy terminus, emulating the bacterial FNR, resulted in an enzyme with decreased affinity for FAD and reduced diaphorase and ferredoxin-dependent cytochrome c reductase activities. The insertion of the ß-hairpin into the corresponding position of the bacterial FNR increased FAD affinity but did not affect its catalytic properties. The same insertion with simultaneous deletion of the carboxy-terminal tryptophan produced a bacterial chimera emulating the plastidic architecture with an increased k(cat) and an increased catalytic efficiency for the diaphorase activity and a decrease in the enzyme's ability to react with its substrates ferredoxin and flavodoxin. Crystallographic structures of the chimeras showed no significant changes in their overall structure, although alterations in the FAD conformations were observed. Plastidic and bacterial FNRs thus reveal differential effects of key structural elements. While the 112-123 ß-hairpin modulates the catalytic efficiency of plastidic FNR, it seems not to affect the bacterial FNR behavior, which instead can be improved by the loss of the C-terminal tryptophan. This report highlights the role of the FAD moiety conformation and the structural determinants involved in stabilizing it, ultimately modulating the functional output of FNRs.

The import of S-adenosylmethionine into the Golgi apparatus is required for the methylation of homogalacturonan.

S-adenosylmethionine (SAM) is the substrate used in the methylation of homogalacturonan (HGA) in the Golgi apparatus. SAM is synthesized in the cytosol, but it is not currently known how it is then transported into the Golgi. In this study, we find that HGA methyltransferase is present in Golgi-enriched fractions and that its catalytic domain faces the lumen of this organelle. This suggests that SAM must be imported into the Golgi. We performed uptake experiments using [methyl-(14)C]SAM and found that SAM is incorporated into the Golgi vesicles, resulting in the methylation of polymers that are sensitive to pectinase and pectin methylesterase but not to proteases. To avoid detecting the transfer reaction, we also used [carboxyl-(14)C]SAM, the uptake of which into Golgi vesicles was found to be sensitive to temperature, detergents, and osmotic changes, and to be saturable with a K(m) of 33 microm. Double-label uptake experiments using [methyl-(3)H]SAM and [carboxyl-(14)C]SAM also revealed a time-dependent increase in the (3)H to (14)C ratio, suggesting that upon transfer of the methyl group, the resulting S-adenosylhomocysteine is not accumulated in the Golgi. SAM incorporation was also found to be inhibited by S-adenosylhomocysteine, whereas UDP-GalA, UDP-GlcA, and acetyl-CoA had no effect. DIDS, a compound that inhibits nucleotide sugar transporters, also had little effect upon SAM incorporation. Interestingly, the combination of UDP-GalA + acetyl-CoA or UDP-GlcA + acetyl-CoA produced a slight increase in the uptake of SAM. These results support the idea that a SAM transporter is required for HGA biosynthesis.