We have analyzed the interaction of DnaK and plant Hsp70 proteins with the wild-type ferredoxin-NADP 1 reductase precursor (preFNR) and mutants containing amino-acid replacements in the targeting sequence. Using an algorithm already developed [Ru Èdiger, S., Germeroth, L., Schneider-Mergener, J. & Bukau, B. (1997) EMBO J. 16, 1501±1507] we observed that 75% of the 727 plastid precursor proteins analyzed contained at least one site with high likelihood of DnaK binding in their transit peptides. Statistical analysis showed a decrease of DnaK binding site frequency within the first 15 amino-acid residues of the transit peptides. Using fusion proteins we detected the interaction of DnaK with the transit peptide of the folded preFNR but not with the mature region of the protein. Discharge of DnaK from the presequence was favored by addition of MgATP. When a putative DnaK binding site was artificially added at the N-terminus of the mature protein, we observed formation of complexes with bacterial and plant Hsp70 molecular chaperones. Reducing the likelihood of DnaK binding by directed mutagenesis of the presequence increased the release of bound DnaK. The Hsp70 proteins from plastids and plant cell cytosol also interacted with the preFNR transit peptide. Overall results are discussed in the context of the proposed models to explain the organelle protein import.

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External flavoprotein monooxygenases comprise a group of flavin-dependent oxidoreductases that catalyze the insertion of one atom of molecular oxygen into an organic substrate and the second atom is reduced to water. These enzymes are involved in a great number of metabolic pathways both in prokaryotes and eukaryotes. Flavoprotein monooxygenases have attracted the attention of researchers for several decades and the advent of recombinant DNA technology caused a great progress in the field. These enzymes are subjected to detailed biochemical and structural characterization and some of them are also regarded as appealing oxidative biocatalysts for the production of fine chemicals and valuable intermediates toward active pharmaceutical ingredients due to their high chemo-, stereo-, and regioselectivity. Here, we review the most representative reactions catalyzed both in vivo and in vitro by prototype flavoprotein monooxygenases, highlighting the strategies employed to produce them recombinantly, to enhance the yield of soluble proteins, and to improve cofactor regeneration in order to obtain versatile biocatalysts. Although we describe the most outstanding features of flavoprotein monooxygenases, we mainly focus on enzymes that were cloned, expressed and used for biocatalysis during the last years.