Potencies of Phosphine Peptide Inhibitors of Mammalian Thimet Oligopeptidase and Neurolysin on Two Bacterial Pz Peptidases

Sugihara, Yusuke; Kawasaki, Akio; Tsujimoto, Yasuhiro; Matsui, Hiroshi; Watanabe, Kunihiko

doi:10.1271/bbb.60534

Cited by 9 publications

(11 citation statements)

References 11 publications

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“…Thus, the tunnel structure of Pz-peptidase A, which belongs to the M3B family, seems more disadvantageous for substrate uptake than the bivalve structure. However, the K m value for Pz-peptidase A is 8.2 M for the substrate 7-methoxycoumarin-3-carboxylyl-Pro-Leu-Gly-Pro-D-Lys(2,4-dinitrophenyl), which is compatible with the figures (8.6 M) for TOP (16,35). The tunnel structure of Pz-peptidase A can function as a gateway for substrates in a comparable manner with that of M3A family enzymes such as TOP.…”

Section: Resultssupporting

confidence: 80%

“…On the other hand, we have found that Pz-peptidase A is sensitive to PPIs, which specifically inhibit TOP and neurolysin (16,36,37) by a similar mechanism and are applicable to x-ray analyses (38). Inhibitors PPI-1 and PPI-2 potently inhibit Pz-peptidase A with K i ϭ 90.1 and 88.9 nM, respectively (16), whereas PPI-2 inhibits neurolysin with K i ϭ 0.4 nM, more strictly and specifically than TOP (K i ϭ 21.0 nM) (36, 37).…”

Section: Resultsmentioning

confidence: 99%

“…Protein Purification and Crystallization-For crystallization, recombinant Pz-peptidase A was purified from an Escherichia coli strain BL21(DE3) harboring plasmid pETA-1 according to methods previously described (16). The purified protein solution, concentrated to ϳ20 mg/ml in 50 mM Tris-HCl (pH 7.5), was incubated in the absence or presence of one of the two PPIs in 12% (w/v) PEG 4000, 0.5 M magnesium acetate, and 0.1 M Tris-HCl (pH 7.0) for 5 days by the hanging-drop vapor diffusion method at 293 K. The PPIs used were benzyloxycarbonyl-Phe-(PO 2 CH 2 )-Ala-Lys-Ser (PPI-1) and GlyPro-Phe-(PO 2 CH 2 )-Gly-Pro-Nle (PPI-2) (gifts from Dr. Vincent Dive), at final concentrations were 0.5 mM (15).…”

Section: Methodsmentioning

confidence: 99%

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The Exquisite Structure and Reaction Mechanism of Bacterial Pz-peptidase A toward Collagenous Peptides

Kawasaki

Nakano

Hosokawa

et al. 2010

Journal of Biological Chemistry

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Pz-peptidase A, from the thermophilic bacterium Geobacillus collagenovorans MO-1, hydrolyzes a synthetic peptide substrate, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-PLGPR), which contains a collagen-specific tripeptide sequence, -Gly-Pro-X-, but does not act on collagen proteins themselves. The mammalian enzyme, thimet oligopeptidase (TOP), which has comparable functions with bacterial Pz-peptidases but limited identity at the primary sequence level, has recently been subjected to x-ray crystallographic analysis; however, no crystal structure has yet been reported for complexes of TOP with substrate analogues. Here, we report crystallization of recombinant Pz-peptidase A in complex with two phosphinic peptide inhibitors (PPIs) that also function as inhibitors of TOP and determination of the crystal structure of these complexes at 1.80 -2.00 Å resolution. The most striking difference between Pz-peptidase A and TOP is that there is no channel running the length of bacterial protein. Whereas the structure of TOP resembles an open bivalve, that of Pz-peptidase A is closed and globular. This suggests that collagenous peptide substrates enter the tunnel at the top gateway of the closed Pz-peptidase A molecule, and reactant peptides are released from the bottom gateway after cleavage at the active site located in the center of the tunnel. One of the two PPIs, PPI-2, which contains the collagen-specific sequence, helped to clarify the exquisite structure and reaction mechanism of Pz-peptidase A toward collagenous peptides. This study describes the mode of substrate binding and its implication for the mammalian enzymes.

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Section: Resultssupporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The Exquisite Structure and Reaction Mechanism of Bacterial Pz-peptidase A toward Collagenous Peptides

Kawasaki

Nakano

Hosokawa

et al. 2010

Journal of Biological Chemistry

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“…The significant positive correlation between altitude and “renin–angiotensin system” was driven by three predicted bacterial proteins that have homologs in diverse organisms including vertebrates, angiotensin I converting enzyme (K01283), prolyl endopeptidase (K01322) and thimet oligopeptidase 1 (K01392) (Supporting Information ). Computational and biochemical studies suggest that these enzymes may have similar functions in bacteria and vertebrates (Kaushik & Sowdhamini, ; Rivière et al., ; Sugihara, Kawasaki, Tsujimoto, Matsui, & Watanabe, ). All three predicted KEGG genes showed positive correlations with altitude in both transects, and two of them had a significant Fisher's combined p ‐value (<0.05) based on Spearman's rho correlations (Supporting Information ).…”

Section: Resultsmentioning

confidence: 99%

Altitudinal variation of the gut microbiota in wild house mice

2018

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The maintenance of oxygen homeostasis in the gut is critical for the maintenance of a healthy gut microbiota. However, few studies have explored how the concentration of atmospheric oxygen affects the gut microbiota in natural populations. Highaltitude environments provide an opportunity to study the potential effects of atmospheric oxygen on the composition and function of the gut microbiota. Here, we characterized the caecal microbial communities of wild house mice (Mus musculus domesticus) in two independent altitudinal transects, one in Ecuador and one in Bolivia, from sea level to nearly 4,000 m. First, we found that differences in altitude were associated with differences in the gut microbial community after controlling for the effects of body mass, diet, reproductive status and population of origin. Second, obligate anaerobes tended to show a positive correlation with altitude, while all other microbes tended to show a negative correlation with altitude. These patterns were seen independently in both transects, consistent with the expected effects of atmospheric oxygen on gut microbes. Prevotella was the most-enriched genus at high elevations in both transects, consistent with observations in high-altitude populations of pikas, ruminants and humans, and also consistent with observations of laboratory mice exposed to hypoxic conditions. Lastly, the renin-angiotensin system, a recently proposed microbiota-mediated pathway of blood pressure regulation, was the top predicted metagenomic pathway enriched in high altitudes in both transects. These results suggest that high-altitude environments affect the composition and function of the gut microbiota in wild mammals. | 2379 SUZUKI et al. S U PP OV RTI N G I N FO R M ATI O NAdditional supporting information may be found online in the Supporting Information section at the end of the article.How to cite this article: Suzuki TA, Martins FM, Nachman MW.Altitudinal variation of the gut microbiota in wild house mice.

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“…The differences in structure and function between the two Pz peptidases, TOP and neurolysin are the focus of our interest at this time. As a first step toward understanding these differences, we found common phosphine inhibitors that showed potent inhibition with K i values in the range 10-100 nM of Pz peptidases as well as TOP and neurolysin (Sugihara et al, 2007). As a second step toward revealing the structures of Pz peptidases, Pz peptidase A was crystallized with one of the inhibitors and a preliminary X ray crystallographic analysis was performed in this study.…”

Section: Introductionmentioning

confidence: 99%

Crystallization and preliminary X-ray crystallographic studies of Pz peptidase A fromGeobacillus collagenovoransMO-1

et al. 2007

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Pz peptidase A is an intracellular M3 metallopeptidase found in the thermophile Geobacillus collagenovorans MO-1 that recognizes collagen-specific tripeptide units (Gly-Pro-Xaa). Pz peptidase A shares common reactions with mammalian thimet oligopeptidase (TOP) and neurolysin, but has extremely low primary sequence identity to these enzymes. In this work, Pz peptidase A was cocrystallized with a phosphine peptide inhibitor (PPI) that selectively inhibits TOP and neurolysin. The crystals belong to space group P2(1), with unit-cell parameters a = 56.38, b = 194.15, c = 59.93 A, beta = 106.22 degrees . This is the first crystallographic study of an M3 family peptidase-PPI complex.

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Potencies of Phosphine Peptide Inhibitors of Mammalian Thimet Oligopeptidase and Neurolysin on Two Bacterial Pz Peptidases

Cited by 9 publications

References 11 publications

The Exquisite Structure and Reaction Mechanism of Bacterial Pz-peptidase A toward Collagenous Peptides

The Exquisite Structure and Reaction Mechanism of Bacterial Pz-peptidase A toward Collagenous Peptides

Altitudinal variation of the gut microbiota in wild house mice

Crystallization and preliminary X-ray crystallographic studies of Pz peptidase A fromGeobacillus collagenovoransMO-1

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