Cleavage of Various Peptides with Pitrilysin from<i>Escherichia coli</i>: Kinetic Analyses Using β-Endorphin and Its Derivatives

Cornista, Joel C.; Ikeuchi, Satoshi; Haruki, Mitsuru; Kohara, Atsuko; Takano, Kazufumi; Morikawa, Masaaki; Kanaya, Shigenori

doi:10.1271/bbb.68.2128

Cited by 17 publications

(15 citation statements)

References 43 publications

(42 reference statements)

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“…The M16A enzyme, pitrilysin, for example, utilizes only one helix and part of the linker region as its hinge (Figures 8, S3). This modification allows a much wider opening of the clamshell than is possible in the dimeric enzymes, such that IDE and pitrilysin can degrade intact insulin, whereas BHP cannot (Cornista et al, 2004; Dabonne et al, 2005). …”

Section: Resultsmentioning

confidence: 99%

Crystal and Solution Structures of a Prokaryotic M16B Peptidase: an Open and Shut Case

et al. 2009

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SUMMARY The M16 family of zinc peptidases comprises a pair of homologous domains that form two halves of a ‘‘clam-shell’’ surrounding the active site. The M16A and M16C subfamilies form one class (‘‘peptidasomes’’): they degrade 30–70 residue peptides, and adopt both open and closed conformations. The eukaryotic M16B subfamily forms a second class (‘‘processing proteases’’): they adopt a single partly-open conformation that enables them to cleave signal sequences from larger proteins. Here, we report the solution and crystal structures of a prokaryotic M16B peptidase, and demonstrate that it has features of both classes: thus, it forms stable ‘‘open’’ homodimers in solution that resemble the processing proteases; but the clam-shell closes upon binding substrate, a feature of the M16A/C peptidasomes. Moreover, clam-shell closure is required for proteolytic activity. We predict that other prokaryotic M16B family members will form dimeric peptidasomes, and propose a model for the evolution of the M16 family.

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Section: Resultsmentioning

confidence: 99%

Crystal and Solution Structures of a Prokaryotic M16B Peptidase: an Open and Shut Case

et al. 2009

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“…ArtI might compensate for C. burnetii arginine auxotrophy [18] by high affinity binding of arginine in what might be a nutrient-limited PV environment. CBU1902 shares homology with Zn metalloendopeptidases, including pitrilysin, an E. coli peptidase that is capable of cleaving numerous substrates [54]. Thus, CBU1902 could modify the PV environment by cleaving harmful acid hydrolases or degrading complex proteinaceous material into peptides/amino acids suitable for transport by C. burnetii.…”

Section: Discussionmentioning

confidence: 99%

Sec-mediated secretion by Coxiella burnetii

et al. 2013

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BackgroundCoxiella burnetii is a Gram-negative intracellular bacterial pathogen that replicates within a phagolysosome-like parasitophorous vacuole (PV) of macrophages. PV formation requires delivery of effector proteins directly into the host cell cytoplasm by a type IVB secretion system. However, additional secretion systems are likely responsible for modification of the PV lumen microenvironment that promote pathogen replication.ResultsTo assess the potential of C. burnetii to secrete proteins into the PV, we analyzed the protein content of modified acidified citrate cysteine medium for the presence of C. burnetii proteins following axenic (host cell-free) growth. Mass spectrometry generated a list of 105 C. burnetii proteins that could be secreted. Based on bioinformatic analysis, 55 proteins were selected for further study by expressing them in C. burnetii with a C-terminal 3xFLAG-tag. Secretion of 27 proteins by C. burnetii transformants was confirmed by immunoblotting culture supernatants. Tagged proteins expressed by C. burnetii transformants were also found in the soluble fraction of infected Vero cells, indicating secretion occurs ex vivo. All secreted proteins contained a signal sequence, and deletion of this sequence from selected proteins abolished secretion. These data indicate protein secretion initially requires translocation across the inner-membrane into the periplasm via the activity of the Sec translocase.ConclusionsC. burnetii secretes multiple proteins, in vitro and ex vivo, in a Sec-dependent manner. Possible roles for secreted proteins and secretion mechanisms are discussed.

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“…First, MP1 shares substrates, but not cleavage sites with other inverzincins. For example, except for the T-R bond in leumorphin, there are no overlapping cleavage sites for this substrate between bacterial pitrilysin (37) and MP1. Second, MP1 exhibits even broader substrate specificity than other M16 metallopeptidases.…”

Section: Discussionmentioning

confidence: 99%

“…Second, MP1 exhibits even broader substrate specificity than other M16 metallopeptidases. This is exemplified by the fact that angiotensin I and bradykinin are not cleaved by protease III (37), insulysin (38) or nardilysin (39), but are substrates of MP1. Additionally, the ability to cleave an N-terminal Phe or Tyr residue is unique to MP1.…”

Section: Discussionmentioning

confidence: 99%

Mammalian Pitrilysin: Substrate Specificity and Mitochondrial Targeting

et al. 2009

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The substrate specificity of the mitochondrial metallopeptidase proteinase 1 (MP1) was investigated and its mitochondrial targeting signal identified. The substrate specificity of MP1 was examined with physiological peptides as substrates. Although the enzyme exhibits broad substrate specificity, there is a trend for peptides containing 13 or more residues to exhibit Km values of 2 μM or less. Three of four peptides containing 11 or fewer residues exhibited Km values above 10 μM. Similarly, peptides containing 13 or more residues exhibited kcat values below 10 min −1 , while three of four peptides containing 11 or fewer residues exhibited kcat values above 30 min −1 . Many of the peptide cleavage sites of MP1 resemble that of the mitochondrial processing protease (MPP), however MP1 does not process the precursor form of citrate synthase. The enzyme does however cleave the released prepeptide from precitrate synthase. A mitochondria localization was shown in MP1 transfected NT2 and HepG2 cells. Deletion of the N-terminal 15 amino acids caused MP1 to be mislocalized to the cytoplasm and nucleus. Furthermore, when fused to GFP, this 15-amino acid N-terminal sequence directed the fusion protein to the mitochondria.Zinc-dependent peptidases of the M16 family possess the active site motif HEXXH in which the two histidine residues coordinate a zinc atom and the glutamate facilitates attack of a water molecule on the scissile bond. A subgroup of the M16 family of metallopeptidases contains an inverted Zn-binding motif HXXEH and these peptidases are referred to as inverzincins (1). The first identified inverzincin was pitrilysin protease III (2), a gene product of the ptr gene in E. coli.Besides protease III (EC 3.4.24.55), several mammalian inverzincins have been identified and classified as M16A family members (3). These include insulysin (insulin degrading enzyme, IDE, EC 3.4.24.56) and nardilysin (NRDc, EC 3.4.24.61). Both protease III and insulysin exhibit a broad substrate specificity cleaving a variety of peptides. The structures of pitrilysin protease III and insulysin (4) are very similar and appear to be "clam-shaped" with the insulysin structure in a closed conformation and the protease III structure in an open conformation. The closed conformation tightly locks down the substrate in the interior of the enzyme, requiring a conformational change to permit product release. This conformational change is likely the Address correspondence to: Louis B. Hersh, Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, B283 BBSRB, 741 South Limestone St., Lexington, Fax: (859) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript ratelimiting step for the insulysin reaction. Both of these enzymes have an active site pocket that can accommodate and interact with a variety of peptides, explaining in part their broad substrate specificity. In contrast, nardilysin has a more defined substrate specificity cleaving peptides primarily at or between dibasic p...

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Cleavage of Various Peptides with Pitrilysin fromEscherichia coli: Kinetic Analyses Using β-Endorphin and Its Derivatives

Cited by 17 publications

References 43 publications

Crystal and Solution Structures of a Prokaryotic M16B Peptidase: an Open and Shut Case

Crystal and Solution Structures of a Prokaryotic M16B Peptidase: an Open and Shut Case

Sec-mediated secretion by Coxiella burnetii

Mammalian Pitrilysin: Substrate Specificity and Mitochondrial Targeting

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