Importance of disulfide bridge formation on folding of phospholipase D from Streptomyces antibioticus

The effects of redox conditions on the folding of phospholipase D (PLD) of Streptomyces antibioticus were investigated. Although the enzyme was very s table even in the presence of 1.0 M guanidinehydrochrolide (Gdn-HCl), the c oexistence of dithiothreitol (DTT) and Gdn-HCl inactivated the enzyme compl etely. The inactivated enzyme recovered its activity by dialysis in which D TT was removed prior to Gdn-HCl, whereas its activity was not recovered whe n Gdn-HCl was removed prior to DTT. In vitro protein synthesis was used for further analyses of the folding process. Active PLD was synthesized In the absence of DTT. The activity increased as the protein synthesis proceeded. In contrast, inactive PLD was synthesized In the presence of DTT. The inac tive PLD could not be effectively activated by simple removal of the reduct ant, while incubation with Gdn-HCl and subsequent removal of DTT followed b y that of Gdn-HCl was a much more effective method for the synthesis of act ive enzymes. From these results, it is suggested that: (i) PLD contains dis ulfide bridge(s), which is (are) necessary for maintaining its tertiary str ucture, (ii) correct formation of the disulfide bridge(s) is a critical ste p in the early stage of the (re)folding process, and (iii) the disulfide br idge(s) further facilitate the folding process, resulting in the synthesis of the active enzymes with the correct structure.