The Arabidopsis thaliana constitutive disease resistance 1 (CDR1) gene product is an aspartic proteinase that has been implicated in disease resistance signaling (Xia, Y., Suzuki, H., Borevitz, J., Blount, J., Guo, Z., Patel, K., Dixon, R. A., and Lamb, C. (2004) EMBO J. 23, 980 -988). This apoplastic enzyme is a member of the group of "atypical" plant aspartic proteinases. As for other enzymes of this subtype, CDR1 has remained elusive until recently as a result of its unusual properties and localization. Here we report on the heterologous expression and characterization of recombinant CDR1, which displays unique enzymatic properties among plant aspartic proteinases. The highly restricted specificity requirements, insensitivity toward the typical aspartic proteinase inhibitor pepstatin A, an unusually high optimal pH of 6.0 -6.5, proteinase activity without irreversible prosegment removal, and dependence of catalytic activity on formation of a homo-dimer are some of the unusual properties observed for recombinant CDR1. These findings unveil a pattern of unprecedented functional complexity for Arabidopsis CDR1 and are consistent with a highly specific and regulated biological function. Plant aspartic proteinases (APs)3 are widely distributed in the plant kingdom and have been detected or purified from monocot and dicot species as well as gymnosperms (1). APs are synthesized as single chain preproenzymes and, upon activation, converted to mature two-chain enzymes. A characteristic feature of "typical" plant AP precursors is the presence of a protein domain of ϳ100 amino acids known as the plant-specific insert, which is highly similar to saposin-like proteins and is removed from the precursors upon activation into the mature form of the enzymes (1).Recently published results on plant APs with unexpected localizations, unusual sequences, and novel structural arrangements (2-10) as well as the identification of a wide variety of AP-like proteins in the Arabidopsis genome (11, 12) have triggered a redefinition of the classification of plant APs. Faro and Gal have recently proposed a new classification that takes into account the diversity of plant APs by grouping them into typical, nucellin-like, and atypical members, with the latter group comprising the majority of the newly identified Arabidopsis APs (11). Despite having low sequence identity, these atypical and nucellin-like APs share several common features such as 1) the absence of the internal segment plant-specific insert in their sequence, 2) an unusually high number of cysteines, 3) the type of amino acids preceding the first catalytic triad, and 4) unexpected localizations, which clearly differentiate them from the well studied typical plant APs. Thus far, only a rather small number of atypical and nucellin-like APs have been studied (2-10). However, the proposed roles in highly regulated processes like plastid homeostasis (tobacco CND41) (9, 10), disease resistance (Arabidopsis CDR1) (7), or programmed cell death (Barley nucellin and Arabidopsis PCS1) (2,...

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The view has been widely held that pepsin-like aspartic proteinases are found only in eukaryotes, and not in bacteria. However, a recent bioinformatics search [Rawlings ND & Bateman A (2009) BMC Genomics 10, 437] revealed that, in seven of 1000 completely sequenced bacterial genomes, genes were present encoding polypeptides that displayed the requisite hallmark sequence motifs of pepsin-like aspartic proteinases. The implications of this theoretical observation prompted us to generate biochemical data to validate this finding experimentally. The aspartic proteinase gene from one of the seven identified bacterial species, Shewanella amazonensis, was expressed in Escherichia coli. The recombinant protein, termed shewasin A, was produced in soluble form, purified to homogeneity, and shown to display properties remarkably similar to those of pepsin-like aspartic proteinases. Shewasin A was maximally active at acidic pH values, cleaving a substrate that has been widely used for assessment of the proteolytic activity of other aspartic proteinases, and displayed a clear preference for cleaving peptide bonds between hydrophobic residues in the P1*P1¢ positions of the substrate. It was completely inhibited by the general inhibitor of aspartic proteinases, pepstatin, and mutation of one of the catalytic Asp residues (in the Asp-Thr-Gly motif of the N-terminal domain) resulted in complete loss of enzymatic activity. It can thus be concluded unequivocally that this Shewanella gene encodes an active pepsin-like aspartic proteinase. It is now beyond doubt that pepsin-like aspartic proteinases are not confined to eukaryotes, but are encoded within some species of bacteria. The distinctions between the bacterial and eukaryotic polypeptides are discussed and their evolutionary relationships are outlined.

Typical aspartic proteinases from plants of the Astereaceae family like cardosins and cyprosins are well-known milk-clotting enzymes. Their effectiveness in cheesemaking has encouraged several studies on other Astereaceae plant species for identification of new vegetable rennets. Here we report on the cloning, expression and characterization of a novel aspartic proteinase precursor from the flowers of Cirsium vulgare (Savi) Ten. The isolated cDNA encoded a protein product with 509 amino acids, termed cirsin, with the characteristic primary structure organization of plant typical aspartic proteinases. The pro form of cirsin was expressed in Escherichia coli and shown to be active without autocatalytically cleaving its pro domain. This contrasts with the acid-triggered autoactivation by pro-segment removal described for several recombinant plant typical aspartic proteinases. Recombinant procirsin displayed all typical proteolytic features of aspartic proteinases as optimum acidic pH, inhibition by pepstatin, cleavage between hydrophobic amino acids and strict dependence on two catalytic Asp residues for activity. Procirsin also displayed a high specificity towards κ-casein and milk-clotting activity, suggesting it might be an effective vegetable rennet. The findings herein described provide additional evidences for the existence of different structural arrangements among plant typical aspartic proteinases.

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