Substrate Binding Changes Conformation of the α-, but Not the β-Subunit of Mitochondrial Processing Peptidase

Gakh, Oleksandr; Obšil, Tomáš; Adamec, Jiřı́; Spížek, J.; Amler, Evžen; Janata, Jiřı́; Kalousek, František

doi:10.1006/abbi.2000.2167

Cited by 13 publications

(11 citation statements)

References 30 publications

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“…Poly-L-lysine was from Sigma-Aldrich. Recombinant yeast MPP was expressed in E. coli and purified as previously described (22). The citrate synthase presequence peptide (MALLTAAARLFGAKNASCLVLAARHAS-NH2) was synthesized by the W.M.…”

Section: Methodsmentioning

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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Section: Methodsmentioning

confidence: 99%

Mammalian Pitrilysin: Substrate Specificity and Mitochondrial Targeting

et al. 2009

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“…The mature form of Yfh1p and the yeast mitochondrial processing peptidase (MPP) were expressed in E. coli and purified as described (Gakh et al, 2001. For expression of wild type CyaY, the cyaY gene was subcloned into the NdeI and HindIII sites of pET24a vector (Novagen).…”

Section: Expression and Purification Of Recombinant Proteinsmentioning

confidence: 99%

Partial conservation of functions between eukaryotic frataxin and theEscherichia colifrataxin homolog CyaY

Bedekovics

Gajdos

Kispál

et al. 2007

FEMS Yeast Research

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Frataxin is a mitochondrial protein structurally conserved from bacteria to humans. Eukaryotic frataxins are known to be involved in the maintenance of mitochondrial iron balance via roles in iron delivery and iron detoxification. The prokaryotic frataxin homolog, CyaY, has been shown to bind and donate iron for the assembly of [2Fe-2S] clusters in vitro. However, in contrast to the severe phenotypes associated with the partial or complete loss of frataxin in humans and other eukaryotes, deletion of the cyaY gene does not cause any obvious alteration of iron balance in bacterial cells, an effect that probably reflects functional redundancy between CyaY and other bacterial proteins. To study CyaY function in a nonredundant setting, we have expressed a mitochondria-targeted form of CyaY in a Saccharomyces cerevisiae strain depleted of the endogenous yeast frataxin protein (yfh1Delta). We show that in this strain CyaY complements to a large extent the loss of iron-sulfur cluster enzyme activities and heme synthesis, and thereby maintains a nearly normal respiratory growth. In addition, CyaY effectively protects yfh1Delta from oxidative damage during treatment with hydrogen peroxide but is less efficient in detoxifying excess labile iron during aerobic growth.

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“…11 Subsequently, we demonstrated a translocation of both the N-and Cterminal domains of α-MPP evoked by the substrate. 19 In this study, measurements were focused on the local dynamics of the conserved GRL of the yeast α-subunit.…”

Section: Discussionmentioning

confidence: 98%

“…Although the roles of each subunit in substrate initial binding are still a matter of contention, most studies have emphasized the relevancy of the α-subunit in substrate binding and recognition. [9][10][11] Similarly to Tom20, MPP specifically recognizes a large variety of diverse mitochondrial presequences that share a low sequence similarity and vary in length, in most cases within the range of 20-50 residues. 6,12,13 Nevertheless, the recognized mitochondrial presequences display common physicochemical properties such as a positive charge and the potential to form an amphiphilic α-helix.…”

Section: Introductionmentioning

confidence: 99%