Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role of the outer-membrane iron transporter FhuA as a potential receptor for the antimicrobial peptide MccJ25 (microcin J25) was studied through a series of in vivo and in vitro experiments. The requirement for both FhuA and the inner-membrane TonB-ExbB-ExbD complex was demonstrated by antibacterial assays using complementation of an fhuA(-) strain and by using isogenic strains mutated in genes encoding the protein complex respectively. In addition, MccJ25 was shown to block phage T5 infection of Escherichia coli, in vivo, by inhibiting phage adhesion, which suggested that MccJ25 prevents the interaction between the phage and its receptor FhuA. This in vivo activity was confirmed in vitro, as MccJ25 inhibited phage T5 DNA ejection triggered by purified FhuA. Direct interaction of MccJ25 with FhuA was demonstrated for the first time by size-exclusion chromatography and isothermal titration calorimetry. MccJ25 bound to FhuA with a 2:1 stoichiometry and a K(d) of 1.2 microM. Taken together, our results demonstrate that FhuA is the receptor for MccJ25 and that the ligand-receptor interaction may occur in the absence of other components of the bacterial membrane. Finally, both differential scanning calorimetry and antimicrobial assays showed that MccJ25 binding involves external loops of FhuA. Unlike native MccJ25, a thermolysin-cleaved MccJ25 variant was unable to bind to FhuA and failed to prevent phage T5 infection of E. coli. Therefore the Val11-Pro16 beta-hairpin region of MccJ25, which is disrupted upon cleavage by thermolysin, is required for microcin recognition.
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PMID:The iron-siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25: role of the microcin Val11-Pro16 beta-hairpin region in the recognition mechanism. 1586 12

The destruction of the Rieske iron-sulfur cluster ([2Fe-2S]) in the bc(1) complex by hematoporphyrin-promoted photoinactivation resulted in the complex becoming proton-permeable. To study further the role of this [2Fe-2S] cluster in proton translocation of the bc(1) complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with mutations at the histidine ligands of the [2Fe-2S] cluster were generated and characterized. These mutants lacked the [2Fe-2S] cluster and possessed no bc(1) activity. When the mutant complex was co-inlaid in phospholipid vesicles with intact bovine mitochondrial bc(1) complex or cytochrome c oxidase, the proton ejection, normally observed in intact reductase or oxidase vesicles during the oxidation of their corresponding substrates, disappeared. This indicated the creation of a proton-leaking channel in the mutant complex, whose [2Fe-2S] cluster was lacking. Insertion of the bc(1) complex lacking the head domain of the Rieske iron-sulfur protein, removed by thermolysin digestion, into PL vesicles together with mitochondrial bc(1) complex also rendered the vesicles proton-permeable. Addition of the excess purified head domain of the Rieske iron-sulfur protein partially restored the proton-pumping activity. These results indicated that elimination of the [2Fe-2S] cluster in mutant bc(1) complexes opened up an otherwise closed proton channel within the bc(1) complex. It was speculated that in the normal catalytic cycle of the bc(1) complex, the [2Fe-2S] cluster may function as a proton-exiting gate.
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PMID:The iron-sulfur cluster of the Rieske iron-sulfur protein functions as a proton-exiting gate in the cytochrome bc(1) complex. 1587 58

The Escherichia coli peptide deformylase (PDF) and Bacillus thermoproteolyticus thermolysin (TLN) are two representative metal-requiring peptidases having remarkably similar active centers but distinctively different metal preferences. Zinc is a competent catalytic cofactor for TLN but not for PDF. Reaction pathways and the associated energetics for both enzymes were determined using combined semiempirical and ab initio quantum mechanical/molecular mechanical modeling, without presuming reaction coordinates. The results confirmed that both enzymes catalyze via the same chemical steps, and reproduced their different preferences for zinc or iron as competent cofactors. Further analyses indicated that different feasibility of the nucleophilic attack step leads to different metal preferences of the two enzymes. In TLN, the substrate is strongly activated and can serve as the fifth coordination ligand of zinc prior to the chemical steps. In PDF, the substrate carbonyl is activated by the chemical step itself, and becomes the fifth coordination partner of zinc only in a later stage of the nucleophilic attack. These leads to a much more difficult nucleophilic attack in PDF than in TLN. Different from some earlier suggestions, zinc has no difficulty in accepting an activated substrate as the fifth ligand to switch from tetra- to penta-coordination in either PDF or TLN. When iron replaces zinc, its stronger interaction with the hydroxide ligand may lead to higher activation barrier in TLN. In PDF, the stronger interactions of iron with ligands allow iron-substrate coordination to take place either before or at a very early stage of the chemical step, leading to effective catalysis. Our calculations also show combined semiempirical and ab initio quantum mechanical modeling can be efficient approaches to explore complicated reaction pathways in enzyme systems.
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PMID:Origins of the different metal preferences of Escherichia coli peptide deformylase and Bacillus thermoproteolyticus thermolysin: a comparative quantum mechanical/molecular mechanical study. 1865 66

The use of nanobiocatalysts, with the combination of nanotechnology and biotechnology, is considered as an exciting and rapidly emerging area. The use of iron oxide magnetic nanoparticles, as enzyme immobilization carriers, has drawn great attention because of their unique properties, such as controllable particle size, large surface area, modifiable surface, and easy recovery. In this study, various gamma-Fe(2)O(3)/Fe(3)O(4) magnetic nanoparticles with immobilized proteases were successfully prepared by three different immobilization strategies including A) direct binding, B) with thiophene as a linker, and C) with triazole as a linker. The oligopeptides syntheses catalyzed by these magnetic nanoparticles (MNPs) with immobilized proteases were systematically studied. Our results show that i) for magnetic nanoparticles immobilized alpha-chymotrypsin, both immobilization strategies A and B furnished good reusability for the Z-Tyr-Gly-Gly-OEt synthesis, the MNPs enzymes can be readily used at least five times without significant loss of its catalytic performance: ii) In the case of Z-Asp-Phe-OMe synthesis catalyzed by magnetic nanoparticles immobilized thermolysin, immobilization Strategy B provided the best recyclability: iii) For the immobilized papain, although Strategy A or B afforded an immobilized enzyme for the first cycle of Z-Ala-Leu-NHNHPh synthesis in good yield, their subsequent catalytic activity decreased rapidly. In general, the gamma-Fe(2)O(3) MNPs were better for use as an immobilization matrix, rather than the Fe(3)O(4) MNPs, owing to their smaller particle size and higher surface area.
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PMID:Protease immobilization on gamma-Fe2O3/Fe3O4 magnetic nanoparticles for the synthesis of oligopeptides in organic solvents. 2044 36

Ovotransferrin is a glycoprotein well-known for its iron-binding property. Ovotransferrin was reported to have antioxidative properties, but the presence of antioxidant peptides within the protein has not been reported. The purpose of the study was to characterize the antioxidant peptides within ovotransferrin. Ovotransferrin was sonicated and hydrolyzed by thermolysin, and peptides from the hydrolysate were fractionated by ion-exchange fast protein liquid chromatography and reversed-phase high-performance liquid chromatography. Fourteen peptides derived from ovotransferrin were characterized using LC-MS/MS, and their oxygen radical absorbance capacity (ORAC) values were determined using synthetic peptides. Two tetrapeptides (Trp-Asn-Ile-Pro and Gly-Trp-Asn-Ile) showed the highest antioxidant activity. Interestingly, the addition of amino acid residues to either the N or C terminus of the two peptides decreased the antioxidant activity, suggesting that the motif of Trp-Asn-Ile is responsible for the high antioxidant activity.
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PMID:Identification of novel antioxidative peptides derived from a thermolytic hydrolysate of ovotransferrin by LC-MS/MS. 2056 71


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