Abstract
Cucumisin is a subtilisin-like serine protease (subtilase) that is found in the juice of melon fruits (Cucumis melo L.). It is synthesized as a preproprotein consisting of a signal peptide, NH(2)-terminal propeptide, and 67-kDa protease domain. We investigated the role of this propeptide (88 residues) in the cucumisin precursor. Complementary DNAs encoding the propeptides of cucumisin, two other plant subtilases (Arabidopsis ARA12 and rice RSP1), and bacterial subtilisin E were expressed in Escherichia coli independently of their mature enzymes. The cucumisin propeptide strongly inhibited cucumisin in a competitive manner with a K(i) value of 6.2 ± 0.55 nm. Interestingly, cucumisin was also strongly inhibited by ARA12 and RSP1 propeptides but not by the subtilisin E propeptide. In contrast, the propeptides of cucumisin, ARA12, and RSP1 did not inhibit subtilisin. Deletion analysis clearly showed that two hydrophobic regions, Asn(32)-Met(38) and Gly(97)-Leu(103), in the cucumisin propeptide were important for its inhibitory activity. Site-directed mutagenesis also confirmed the role of a Val(36)-centerd hydrophobic cluster within the Asn(32)-Met(38) region in cucumisin inhibition. Circular dichroism spectroscopy revealed that the cucumisin propeptide had a secondary structure without a cognate protease domain and that the thermal unfolding of the propeptide at 90 °C was only partial and reversible. A tripeptide, Ile(35)-Val(36)-Tyr(37), in the Asn(32)-Met(38) region was thought to contribute toward the formation of a proper secondary structure necessary for cucumisin inhibition. This is the first report on the function and structural information of the propeptide of a plant serine protease.
Highlights
Ase sequences corresponding to almost 3% of the proteome, representing all five catalytic types: serine, cysteine, aspartic acid, metallo, and threonine [1, 2]
The primary structure of cucumisin deduced from the cDNA sequence revealed that it is synthesized as a precursor consisting of four functional domains: a possible signal peptide (22 amino acid residues); NH2-terminal prosequence (88 residues); 54-kDa protease domain (505 residues), which is the active enzyme domain of the 67-kDa native cucumisin; and 14-kDa COOHterminal polypeptide (116 residues), which arises by limited autolysis of the 67-kDa native cucumisin [3, 5]
The sequences of plant subtilase propeptides had low identities (Ͻ20%) with that of subtilisin E, they contained conserved motifs N1 and N2, which were previously identified within bacterial subtilisins and appeared to be critical for protease domain folding [26]
Summary
Stress-induced processing of a membrane-associated transcription factor, inducing the expression of stress response gene [12]. Amino acids at positions ϩ1 and ϩ2 are both Thr, and those at positions ϩ3 and ϩ4 are Arg/His and Thr/Ser, respectively [14] This suggests a common mechanism for the propeptide processing of plant subtilase precursors. Detailed mechanisms of subsequent processing and activation of plant subtilase precursors are unknown except for a recent report demonstrating that the prodomain cleavage of a tomato subtilase 3 (SlSBT3) occurs autocatalytically and that the zymogen maturation is an intramolecular process [15]. The prodomains of bacterial subtilisins are autocatalytically cleaved at their junction with the catalytic domains They remain non-covalently bound and act as specific inhibitors of proteolytic activity [16, 17]. This is the first report demonstrating that the propeptide of a plant serine protease acts as a tightly binding competitive inhibitor of the mature enzyme and that the secondary structure of the propeptide is indispensable for its inhibitory activity
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
