Cyanobacterial NAD(P) + -reducing reversible hydrogenases comprise five subunits. Four of them (HoxF, HoxU, HoxY, and HoxH) are also found in the well-described related enzyme from Ralstonia eutropha. The fifth one (HoxE) is not encoded in the R. eutropha genome, but shares homology with the N-terminal part of R. eutropha HoxF. However, in cyanobacteria, HoxE contains a 2Fe-2S cluster-binding motif that is not found in the related R. eutropha sequence. In order to obtain some insights into the role of HoxE in cyanobacteria, we deleted this subunit in Synechocystis PCC6803. Three types of interaction of the cyanobacterial hydrogenase with pyridine nucleotides were tested: (a) reductive activation of the NiFe site, for which NADPH was found to be more efficient than NADH; (b) H 2 production, for which NADH appeared to be a more efficient electron donor than NADPH; and (c) H 2 oxidation, for which NAD + was a much better electron acceptor than NADP + . Upon hoxE deletion, the Synechocystis hydrogenase active site remained functional with artificial electron donors or acceptors, but the enzyme became unable to catalyze H 2 production or uptake with NADH ⁄ NAD + . However, activation of the electron transfer-independent H ⁄ D exchange reaction by NADPH was still observed in the absence of HoxE, whereas activation of this reaction by NADH was lost. These data suggest different mechanisms for diaphorase-mediated electron donation and catalytic site activation in cyanobacterial hydrogenase.

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Structure similarity searches using a combinatorial extension approach revealed that a protein fold structurally related to the sphingolipid binding domain (SBD) of HIV-1 gp120 (V3 loop) is present on pancreatic bile salt-dependent lipase (BSDL). A synthetic peptide derived from the predicted V3-like domain of BSDL interacted with reconstituted monolayers of sphingolipids such as GalCer and GlcCer. Using Chinese hamster ovary cells stably transfected with the cDNA encoding the rat BSDL (CHO-3B clone) or pancreatic SOJ-6 cells expressing the human BSDL as models, we showed that the enzyme cofractionates with caveolin-1. The secretion of BSDL by CHO-3B cells was inhibited by permeable drugs affecting rafts structure (D609, PDMP, and filipin). Data suggest that the functional interaction between the BSDL SBD and lipid rafts is physiologically relevant and could be essential for sensing the BSDL folding prior to secretion. A tentative model accounting for the phosphorylation-induced dissociation of BSDL from rafts is presented.