Exploring the subsite‐structure of vimelysin and thermolysin using FRETS‐libraries

Oda, Kōhei; Takahashi, Toshihiro; Takada, Katsumi; Tsunemi, Masahiko; Ng, Kenneth; Hiraga, Kazumi; Harada, Shigeharu

doi:10.1016/j.febslet.2005.07.089

Cited by 17 publications

(8 citation statements)

References 17 publications

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“…Among M4 and M13 gluzincins, the S1′ specificity residues form a pocket that is the chief determinant of substrate specificity . In thermolysin (M4) and neprilysin (M13) X‐ray crystal structures, S1′ specificity pockets are composed of aromatic and hydrophobic residues favoring substrate P1′ side chains that possess complimentary hydrophobic character.…”

Section: Discussionsupporting

confidence: 58%

The tripartite architecture of the eukaryotic integral membrane protein zinc metalloprotease Ste24

2019

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Ste24 enzymes, a family of eukaryotic integral membrane proteins, are zinc metalloproteases (ZMPs) originally characterized as “CAAX proteases” targeting prenylated substrates, including a‐factor mating pheromone in yeast and prelamin A in humans. Recently, Ste24 was shown to also cleave nonprenylated substrates. Reduced activity of the human ortholog, HsSte24, is linked to multiple disease states (laminopathies), including progerias and lipid disorders. Ste24 possesses a unique “α‐barrel” structure consisting of seven transmembrane (TM) α‐helices encircling a large intramembranous cavity (~14 000 Å3). The catalytic zinc, coordinated via a HExxH…E/H motif characteristic of gluzincin ZMPs, is positioned at one of the cavity's bases. The interrelationship between Ste24 as a gluzincin, a long‐studied class of soluble ZMPs, and as a novel cavity‐containing integral membrane protein protease has been minimally explored to date. Informed by homology to well‐characterized soluble, gluzincin ZMPs, we develop a model of Ste24 that provides a conceptual framework for this enzyme family, suitable for development and interpretation of structure/function studies. The model consists of an interfacial, zinc‐containing “ZMP Core” module surrounded by a “ZMP Accessory” module, both capped by a TM helical “α‐barrel” module of as yet unknown function. Multiple sequence alignment of 58 Ste24 orthologs revealed 38 absolutely conserved residues, apportioned unequally among the ZMP Core (18), ZMP Accessory (13), and α‐barrel (7) modules. This Tripartite Architecture representation of Ste24 provides a unified image of this enzyme family.

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Section: Discussionsupporting

confidence: 58%

The tripartite architecture of the eukaryotic integral membrane protein zinc metalloprotease Ste24

2019

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“…Substrate specificity of the proteinase A novel type of fluorescence energy transfer (FRET) combinatorial library, which has been proven to be a sensitive and reliable substrate for determination of the substrate specificity of proteinases, 14,22,23) was used in order to determine the substrate specificity of the purified proteinase from Halobacillus sp. SR5-3.…”

Section: Amino-terminal Sequence Analysismentioning

confidence: 99%

A Halophilic Serine Proteinase fromHalobacillussp. SR5-3 Isolated from Fish Sauce: Purification and Characterization

Namwong

Hiraga

Takada

et al. 2006

Bioscience, Biotechnology, and Biochemistry

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A halophilic bacterium was isolated from fish sauce, classified, and named Halobacillus sp. SR5-3. A purified 43-kDa proteinase produced by this bacterium showed optimal activity at 50 degrees C and pH 9-10 in 20% NaCl. The activity of the enzyme was enhanced about 2.5-fold by the addition of 20-35% NaCl, and the enzyme was highly stabilized by NaCl. It was found to be a serine proteinase related to either chymotrypsin or subtilisin. It absolutely preferred Ile at the P(2) position of substrates. Thus, the enzyme was found to be a halophilic serine proteinase with unique substrate specificity.

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“…Studies of cathepsin K showed that its unique collagenolytic activity among cathepsins is caused by a preference for arginine and lysine at the P1 position and proline and glycine at the P2 and P3 positions [28]; neither of these residues, especially proline, is preferred by other human cathepsins. Previously, we have studied the residue preferences in SOT and SGT using fluorescence energy transfer substrate (FRETS) combinatorial libraries [29–31], which consist of a total of 475 peptide substrates. In contrast with other studies, we found that the P1, P2 and P3 preferences of SOT resembled those of SGT.…”

Section: Resultsmentioning

confidence: 99%

The role of Tyr71 in Streptomyces trypsin on the recognition mechanism of structural protein substrates

Uesugi¹,

Usuki²,

Iwabuchi³

et al. 2009

The FEBS Journal

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Studies of substrate recognition by serine proteases have focused on specificities at the primary S1-Sn sites, but topological specificities (i.e. recognition at distinct three-dimensional structural motifs) have not been established. This is the first report to identify the key amino acid residue conferring topological specificity. A serine protease from Streptomyces omiyaensis (SOT), which is a trypsin-like enzyme, was chosen as a model enzyme to clarify the recognition mechanism of structural protein substrates in serine proteases. We have found previously that the topological specificities of SOT and S. griseus trypsin (SGT) for high molecular mass substrates differ greatly, even though the enzymes have similar primary structures. In this study, we constructed chimeras between SOT and SGT using an in vivo DNA shuffling system and several mutants to identify the key residues involved in topological specificities. By comparing the substrate specificities of chimeras and mutants, we found that residue 71 of SOT, which is separate from the catalytic triad, contributes to the topological specificity. Using site-directed mutagenesis, residue 71 of SOT was also found to be crucial for catalytic efficiency and enzyme conformation.

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Exploring the subsite‐structure of vimelysin and thermolysin using FRETS‐libraries

Cited by 17 publications

References 17 publications

The tripartite architecture of the eukaryotic integral membrane protein zinc metalloprotease Ste24

The tripartite architecture of the eukaryotic integral membrane protein zinc metalloprotease Ste24

A Halophilic Serine Proteinase fromHalobacillussp. SR5-3 Isolated from Fish Sauce: Purification and Characterization

The role of Tyr71 in Streptomyces trypsin on the recognition mechanism of structural protein substrates

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