The folding and activity of the extracellular lipase of Rhizopus oryzae are modulated by a prosequence.

The fungus Rhizopus oryzae synthesizes an extracellular lipase precursor bearing N-terminal pre- and pro-sequences. Our studies in Escherichia coli and using recombinant lipase in vitro indicate that the prosequence of 97 amino acids has at least two functions. First, it modulates the enzyme activity of the lipase so that this enzyme can initially be synthesized in a non-destructive form. Direct synthesis of the mature form of the lipase in the cell has toxic consequences, at least partly because of phospholipase activity that is suppressed in the proprotein. Secondly, it supports folding of the lipase via a pathway influenced by a single cysteine residue at position - 68. Mutational analysis of the prosequence demonstrates not only the key role of this cysteine residue but also the importance of the neighbouring amino acids. In particular, Arg-69 probably enhances the leaving group character of Cys-68. We propose a model in which Cys-68 acts as an intramolecular thiodisulphide reagent, playing a catalytic role in the folding of the enzyme. The prosequence is capable of performing the described functions both in cis and in trans.