Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease.
Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones.
From the Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia and.
Raisa A
RA
Galiullina
Division of Bioorganic Chemistry, Department of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland.
Paulina
P
Kasperkiewicz
From the Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia and.
Nina V
NV
Chichkova
Division of Bioorganic Chemistry, Department of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland.
Aleksandra
A
Szalek
From the Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia and.
Marina V
MV
Serebryakova
Division of Bioorganic Chemistry, Department of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland.
Marcin
M
Poreba
Division of Bioorganic Chemistry, Department of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland marcin.drag@pwr.edu.pl.
Marcin
M
Drag
From the Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia and varta@genebee.msu.ru.
Andrey B
AB
Vartapetian
D000595:Amino Acid Sequence; D001665:Binding Sites; D016923:Cell Death; D002766:Cholecystokinin / Q000378:metabolism; D005755:Gastrins / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D012275:Oryza / Q000166:cytology / Q000201:enzymology*; D011485:Protein Binding; D013379:Substrate Specificity; D020860:Subtilisin / Q000737:chemistry / Q000378:metabolism*
D005755:Gastrins; D002766:Cholecystokinin; D020860:Subtilisin
aspartate (aspartic acid); cell death; cholecystokinin; gastrin; peptide hormone; phytaspase; proteolysis; proteolytic enzyme; substrate specificity