Isolation and Characterization of a Possible Native Cucumisin from Developing Melon Fruits and Its Limited Autolysis to Cucumisin

Yamagata, Hiroshi; Ueno, Shuji; Iwasaki, Teruo

doi:10.1080/00021369.1989.10869404

Cited by 22 publications

(25 citation statements)

References 6 publications

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“…The biosynthesis of ethylene in melon fruit occurs at a late stage in fruit development, and the level of 1-aminocyclopropane-1-carboxylate oxidase, a key ethylene biosynthetic enzyme, is very low in unripe melon fruit (18,19). Synthesis of cucumisin, however, was observed at a very early stage (about 5 DAP) of fruit development before the onset of ripening (6,7). Also, ethylene-responsive elements such as the GCC box conserved in the 5Ј upstream region of many ethylene-inducible genes (20,21) could not be found in the cucumisin promoter (Fig.…”

Section: Discussionmentioning

confidence: 99%

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TGTCACA Motif Is a Novel cis-Regulatory Enhancer Element Involved in Fruit-specific Expression of the cucumisin Gene

Yamagata

Yonesu

Hirata

et al. 2002

Journal of Biological Chemistry

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Cucumisin, a subtilisin-like serine protease, is expressed at high levels in the fruit of melon (Cucumis melo L.) and accumulates in the juice. We investigated roles of the promoter regions and DNA-protein interactions in fruit-specific expression of the cucumisin gene. In transient expression analysis, a chimeric gene construct containing a 1.2-kb cucumisin promoter fused to a ␤-glucuronidase (GUS) reporter gene was expressed in fruit tissues at high levels, but the promoter activities in leaves and stems were very low. Deletion analysis indicated that a positive regulatory region is located between nucleotides ؊234 and ؊214 relative to the transcriptional initiation site. Gain-of-function experiments revealed that this 20-bp sequence conferred fruit specificity and contained a regulatory enhancer. Gel mobility shift experiments demonstrated the presence of fruit nuclear factors that interact with the cucumisin promoter. A typical G-box (GACACGTGTC) present in the 20-bp sequence did not bind fruit protein, but two possible cis-elements, an I-box-like sequence (AGATAT-GATAAAA) and an odd base palindromic TGTCACA motif, were identified in the promoter region between positions ؊254 and ؊215. The I-box-like sequence bound more tightly to fruit nuclear protein than the TGTCACA motif. The I-box-like sequence functions as a negative regulatory element, and the TGTCACA motif is a novel enhancer element necessary for fruit-specific expression of the cucumisin gene. Specific nucleotides responsible for the binding of fruit nuclear protein in these two elements were also determined.Timing and levels of gene expression are critical to the proper development of eukaryotic organisms. Regulation of the expression pattern of a particular gene can involve the specific binding of trans-acting factors to the cognate cis-elements, constituting a crucial step in transcriptional initiation and, in turn, on the spatial and temporal expression of genes. Plant genes that show tissue specificity, developmental specificity, and a wide range of expression levels have been characterized, whereas their expression patterns are also influenced by environmental stimuli. A family of genes for fruit proteins provide a model system for the study of the regulatory mechanisms of plant genes, since their expression is restricted to a specific tissue and stage during fruit development (1). A number of fruit-specific genes that are activated during ripening have been isolated from tomato and other fruits, and genes responding to ethylene and nonethylene signals have been identified (1, 2). The promoters of fruit-specific genes would also be of great interest for use in strategies to manipulate fruit metabolism and produce valuable proteins such as antibody, biopharmaceuticals, and edible vaccines through methods of genetic engineering (3-5). However, the detailed mechanisms by which the expression of fruit protein genes are regulated are poorly understood, as many of the essential cis-elements have not been identified.Melon cucumisin, an extracellular...

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Section: Discussionmentioning

confidence: 99%

“…Melon cucumisin, an extracellular subtilisin-like serine protease, is expressed at high levels in fruit, where it comprises more than 10% of the total protein (6). No expression is detected in other parts of the melon plant.…”

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TGTCACA Motif Is a Novel cis-Regulatory Enhancer Element Involved in Fruit-specific Expression of the cucumisin Gene

Yamagata

Yonesu

Hirata

et al. 2002

Journal of Biological Chemistry

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“…It comprises more than 10% of the total juice protein and is synthesized in the central parts of the fruits (3). Cucumisin is synthesized and accumulated only in melon fruits, and a cis-regulatory enhancer element in the cucumisin promoter regulates fruit-specific expression of the cucumisin gene (4).…”

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Functional Analysis of the Cucumisin Propeptide as a Potent Inhibitor of Its Mature Enzyme

Nakagawa

Ueyama

Tsuruta

et al. 2010

Journal of Biological Chemistry

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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. 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.Proteases play key roles in diverse processes regulating plant growth, development, and responses to environmental stimuli. They are necessary for protein turnover, strict protein quality control, and degrading specific sets of proteins. Comparative genomics analyses could provide valuable insights into the abundance and roles of various plant protease families. For example, the Arabidopsis thaliana genome has over 550 protease sequences corresponding to almost 3% of the proteome, representing all five catalytic types: serine, cysteine, aspartic acid, metallo, and threonine (1, 2). Of these, serine proteases appear to be the largest class of plant proteases, although protease activity has been demonstrated only by a few of them.Cucumisin (EC 3.4.21.25) is an extracellular thermostable alkaline serine protease that is expressed at high levels in melon fruits (Cucumis melo L.). It comprises more than 10% of the total juice protein and is synthesized in the central parts of the fruits (3). Cucumisin is synthesized and accumulated only in melon fruits, and a cis-regulatory enhancer element in the cucumisin promoter regulates fruit-specific expression of the cucumisin gene (4). We have determined the complete nucleotide sequence of a cucumisin cDNA, the first sequenced plant serine protease, and found that cucumisin is a member of the subtilisin (EC 3.4.21.62) superfamily characterized by a catalytic triad of three amino acids: Asp, His, and Ser (5). 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); NH 2 -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 optimal...

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“…The present study was conducted to investigate the enzyme extract and to identify metals, content in Sudanese S. dubium seed (27 …”

Section: Resultsmentioning

confidence: 99%

“…The temperature profile of the enzyme was agreed with those of subtilisin/cucumisin likeplant serine proteases reported by AsifUllah et al In conclusion, compared to other purification procedures done we concluded that a simple purification procedure has been developed in this study to obtain a very active and stable enzyme from S. dubium seeds for milk-clotting. However, further purification is requiredto get a highly pure enzyme (11,14,27).…”

Section: Effect Of Ph On Partially Purified Enzyme Activitymentioning

confidence: 99%

Evaluation of Minerals Content in SolaniumDubium Collected From White Nilein Central Sudan

Mohammed¹

2017

IOSR JAC

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Abstract:Some plants of the family solanaceae such as Solanumdubium"Gubbain", (20-90 °C). The results of substrate specificity of the enzyme showed that the partially purified enzyme preferred both hydrophilic and hydrophobic amino acid residues at P1 position. The catalytic efficiency of the purified enzyme was enhanced by an aliphatic amino acids (Leu) compared to aromatic residue (Phe) at P1 position at the same site.And the morphology of solanum seeds was studied with scanning electron microscopy of mucilage at different magnification using Philips, Lancashire,6,7). Also the fractionation of structure was observed with infera red spectroscopy in figure (8). In this study some metals was determined like Mn , Cr, Cd , Zn. , Fe, and basic element like Ca ,Mg , K , P

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Isolation and Characterization of a Possible Native Cucumisin from Developing Melon Fruits and Its Limited Autolysis to Cucumisin

Cited by 22 publications

References 6 publications

TGTCACA Motif Is a Novel cis-Regulatory Enhancer Element Involved in Fruit-specific Expression of the cucumisin Gene

TGTCACA Motif Is a Novel cis-Regulatory Enhancer Element Involved in Fruit-specific Expression of the cucumisin Gene

Functional Analysis of the Cucumisin Propeptide as a Potent Inhibitor of Its Mature Enzyme

Evaluation of Minerals Content in SolaniumDubium Collected From White Nilein Central Sudan

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