Crystal Structure of Cucumisin, a Subtilisin-Like Endoprotease from Cucumis melo L.

Murayama, Kazutaka; Kato-Murayama, M.; Hosaka, Toshiaki; Sotokawauchi, Ami; Yokoyama, Shigeyuki; Arima, Kazunari; Shirouzu, Mikako

doi:10.1016/j.jmb.2012.07.013

Cited by 29 publications

(41 citation statements)

References 30 publications

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“…Consistent with this notion, dimerization was not observed in the second structurally characterized plant subtilase, cucumisin from melon fruits (17). In cucumisin, the PA domain is located comparatively close to the active site channel and appears to contribute to substrate selectivity (17).…”

mentioning

confidence: 78%

“…SBT3 differs from other SBTs as it is active as a homodimer, in which an autoinhibitory ␤-hairpin is immobilized by the PA domain (15). Bacterial and mammalian SBTs lack the PA domain, and in cucumisin the PA domain is not involved in dimerization but may rather contribute to substrate selectivity (17). These differences will affect the interaction of the PP with the active site of the enzyme and are likely to account for the unique specificity of the SBT3-SBT3PP interaction.…”

Section: Discussionmentioning

confidence: 99%

“…However, structural modeling of representative Arabidopsis SBTs indicated that PA domain-mediated dimerization as an autoregulatory mechanism for enzyme activation is unlikely to be a general property of all plant SBTs (16). Consistent with this notion, dimerization was not observed in the second structurally characterized plant subtilase, cucumisin from melon fruits (17). In cucumisin, the PA domain is located comparatively close to the active site channel and appears to contribute to substrate selectivity (17).…”

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confidence: 95%

“…Additional distinguishing features of SBT3 and cucumisin as compared with bacterial SBTs include a stabilizing fibronectin III-like domain at the C terminus and the lack of bound calcium (15,17). Two Ca 2ϩ binding sites, one of high and the other of low affinity, are typically found in S8A subtilases (3), and the binding of calcium ions contributes to enzyme stability (18).…”

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confidence: 99%

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Functional Characterization of Propeptides in Plant Subtilases as Intramolecular Chaperones and Inhibitors of the Mature Protease

Meyer

Leptihn

Welz

et al. 2016

Journal of Biological Chemistry

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Subtilisin-like serine proteases (SBTs) are extracellular proteases that depend on their propeptides for zymogen maturation and activation. The function of propeptides in plant SBTs is poorly understood and was analyzed here for the propeptide of tomato subtilase 3 (SBT3PP). SBT3PP was found to be required as an intramolecular chaperone for zymogen maturation and secretion of SBT3 in vivo. Secretion was impaired in a propeptide-deletion mutant but could be restored by co-expression of the propeptide in trans. SBT3 was inhibited by SBT3PP with a K d of 74 nM for the enzyme-inhibitor complex. With a melting point of 87°C, thermal stability of the complex was substantially increased as compared with the free protease suggesting that propeptide binding stabilizes the structure of SBT3. Even closely related propeptides from other plant SBTs could not substitute for SBT3PP as a folding assistant or autoinhibitor, revealing high specificity for the SBT3-SBT3PP interaction. Separation of the chaperone and inhibitor functions of SBT3PP in a domain-swap experiment indicated that they are mediated by different regions of the propeptide and, hence, different modes of interaction with SBT3. Release of active SBT3 from the autoinhibited complex relied on a pH-dependent cleavage of the propeptide at Asn-38 and Asp-54. The remarkable stability of the autoinhibited complex and pH dependence of the secondary cleavage provide means for stringent control of SBT3 activity, to ensure that the active enzyme is not released before it reaches the acidic environment of the trans-Golgi network or its final destination in the cell wall. Subtilases (SBTs)2 are found in all kingdoms of life. They constitute the S8 family of serine peptidases (1) and are characterized by a specific arrangement of the aspartate, histidine, and serine residues of the catalytic triad within the active site of the enzyme (2). SBTs include general proteases with relaxed substrate specificity for protein degradation and turnover as well as processing enzymes that cleave selected substrates at highly specific sites (3). Most bacterial SBTs are of the catabolic type including subtilisin E and subtilisin Carlsberg from Bacillus subtilis and Bacillus licheniformis, the prototypical members of the S8A subfamily of subtilases (4). The type-example for subfamily S8B is kexin from Saccharomyces cerevisiae, which was identified as the first SBT from eukaryotes and the first with narrow specificity for dibasic cleavage sites (5). Dibasic cleavage specificity is typically found also in the seven kexin-related proprotein convertases (PCs) in mammals (6). In contrast, all plant SBTs belong to subfamily S8A. They are thus more closely related to bacterial subtilisins than to kexin, but they comprise both, general proteases for protein turnover as well as processing enzymes for limited proteolysis at highly specific sites (7,8).Early structural investigations of subtilisin E showed that it is produced with an N-terminal signal peptide targeting the protein to the periplasmic spac...

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confidence: 78%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 95%

mentioning

confidence: 99%

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Functional Characterization of Propeptides in Plant Subtilases as Intramolecular Chaperones and Inhibitors of the Mature Protease

Meyer

Leptihn

Welz

et al. 2016

Journal of Biological Chemistry

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“…The presence of the LMW band could be due to the degradation of the protease or a C-terminal trimming of mature phytaspase, as is common in most plant subtilases, though this has not been confirmed in the present study. 8,10,20,21 The bands equivalent to 26 kDa in the wash and eluted fractions corresponded to free GST tag. The appearance of the »69 kDa bands in the 'wash' as well as the eluted fractions may have occurred due to autocatalytic cleavage of the 'pro' domain from pre-prophytaspase releasing mature phytaspase.…”

Section: Purification and Autoprocessing Of Gst-pre-prophytaspasementioning

confidence: 99%

Tobacco phytaspase: Successful expression in a heterologous system

Narayanan¹,

Sanpui

Sahoo³

et al. 2017

Bioengineered

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Phytaspase, a plant serine protease, has been demonstrated to play an important role in the programmed cell death of various plants. Phytaspase is synthesized as an inactive proenzyme containing an N-terminal signal peptide followed by a pro-domain and a mature protease catalytic domain. Pre-prophytaspase autocatalytically processes itself into a pro-domain and an active mature phytaspase enzyme. We have recently demonstrated the successful expression of mature phytaspase from tobacco in a bacterial system. Herein, we focus on the expression of preprophytaspase as a GST-tag fusion and on its purification by affinity chromatography.

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Sequence analysis and crystal structure of a glycosylated protease from Euphorbia resinifera latex for its proteolytic activity aspect

Siritapetawee

Attarataya

Charoenwattanasatien

2022

Biotech and App Biochem

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The investigation of a plant glycosylated serine protease (EuRP-61) isolated from Euphorbia resinifera latex for potential antiplatelet and anticoagulation activities has been previously reported. In the present study, the protein sequence and native crystal structure of EuRP-61 were characterized. The structure was identified using single-wavelength anomalous diffraction with a refinement resolution of 1.7 Å (PDB ID: 7EOX). The main structural components of EuRP-61 were composed of three domains: catalytic, protease-associated (PA), and fibronectin type III (Fn3)-like domains. The crystal structure revealed that some loops in the PA and catalytic domains of EuRP-61 were different from the other subtilisinlike proteases (cucumisin and SBT3). These different loops might be involved in the general monomer formation of EuRP-61, substrate specificity, and maintenance of the catalytic domain. The Fn3-like domain may provide flexibility to the enzyme to bind with various substrates and cell receptors. Additionally, the active site of EuRP-61 consisted of the catalytic triad of Ser434, His106, and Asp32, similar to other serine proteases. The present study provides additional information and insight into the protease and antithrombotic activities of EuRP-61, which could contribute to further development of this enzyme for biomedical treatment.

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Crystal Structure of Cucumisin, a Subtilisin-Like Endoprotease from Cucumis melo L.

Cited by 29 publications

References 30 publications

Functional Characterization of Propeptides in Plant Subtilases as Intramolecular Chaperones and Inhibitors of the Mature Protease

Functional Characterization of Propeptides in Plant Subtilases as Intramolecular Chaperones and Inhibitors of the Mature Protease

Tobacco phytaspase: Successful expression in a heterologous system

Sequence analysis and crystal structure of a glycosylated protease from Euphorbia resinifera latex for its proteolytic activity aspect

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