Structural features of plant subtilases

Rose, Rolf; Schaller, Andreas; Ottmann, C.

doi:10.4161/psb.5.2.11069

Cited by 35 publications

(37 citation statements)

References 23 publications

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“…X-ray structure analysis of SBT3 from tomato revealed that the PA domain mediates homo-dimerization via interaction with an unusual ␤-hairpin that is not found in bacterial SBTs resulting in the activation of the tomato enzyme (15). 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).…”

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confidence: 99%

“…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). Despite the lack of calcium, both SBT3 and cucumisin exhibit remarkable thermal stability indicating that plants evolved different means to stabilize the subtilisin fold (8,15,16). The prodomain was not included in the crystal structure of SBT3 or cucumisin, and its function in plant SBTs is thus still poorly understood.…”

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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: 99%

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confidence: 99%

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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“…1,5 Subtilisin-like serine proteases (S8 family), commonly termed subtilases, are the second most ubiquitous serine proteases and can be found in bacteria, eukaryotes and even viruses. 6,7 Subtilases possess the same catalytic triad found in chymotrypsin, differing only in arrangement; Asp-His-Ser is found in subtilases, and His-Asp-Ser is found in chymotrypsin. Approximately 200 subtilases have been discovered, and they have been categorized into 6 families based on sequence homology: subtilisin, thermitase, proteinase K, lantibiotic peptidase, kexin and pyrolysin.…”

Section: Phytaspase Is a Member Of The Plant Subtilase Familymentioning

confidence: 99%

“…Subtilases are synthesized as zymogens. 7,8 They contain an N-terminal signal peptide followed by a prodomain and then a C-terminal catalytic domain (mature protease domain). The zymogen undergoes maturation to the mature protease by autocatalytically cleaving the peptide bond between the pro-domain and the mature protease domain.…”

Section: Phytaspase Is a Member Of The Plant Subtilase Familymentioning

confidence: 99%

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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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