Interactions of a Novel Inhibitor from an ExtremophilicBacillus sp. with HIV-1 Protease

Dash, Chandravanu; Rao, M. R. Raghavendra

doi:10.1074/jbc.m005662200

Cited by 22 publications

(29 citation statements)

References 40 publications

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“…The amino acid sequence of ATBI was determined to be Ala-GlyLys-Lys-Asp-Asp-Asp-Asp-Pro-Pro-Glu (13). Searches of the protein databases have failed to identify any antifungal proteins with significant homology to ATBI.…”

Section: Resultsmentioning

confidence: 99%

Novel Bifunctional Inhibitor of Xylanase and Aspartic Protease: Implications for Inhibition of Fungal Growth

Dash

Ahmad

Nath

et al. 2001

Antimicrob Agents Chemother

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A novel bifunctional inhibitor (ATBI) from an extremophilic Bacillus sp. exhibiting an activity against phytopathogenic fungi, including Alternaria, Aspergillus, Curvularia, Colletotricum, Fusarium, and Phomopsis species, and the saprophytic fungus Trichoderma sp. has been investigated. The 50% inhibitory concentrations of ATBI ranged from 0.30 to 5.9 g/ml, whereas the MIC varied from 0.60 to 3.5 g/ml for the fungal growth inhibition. The negative charge and the absence of periodic secondary structure in ATBI suggested an alternative mechanism for fungal growth inhibition. Rescue of fungal growth inhibition by the hydrolytic products of xylanase and aspartic protease indicated the involvement of these enzymes in cellular growth. The chemical modification of Asp or Glu or Lys residues of ATBI by 2,4,6-trinitrobenzenesulfonic acid and Woodward's reagent K, respectively, abolished its antifungal activity. In addition, ATBI also inhibited xylanase and aspartic protease competitively, with K i values 1.75 and 3.25 M, respectively. Our discovery led us to envisage a paradigm shift in the concept of fungal growth inhibition for the role of antixylanolytic activity. Here we report for the first time a novel class of antifungal peptide, exhibiting bifunctional inhibitory activity.The primary current means for the identification of new antifungal agents are represented by screening of the vast biodiversity prevalent in natural resources such as soil samples, marine waters, insects, and tropical plants (6,8). The need for safe and effective antifungal agents has triggered considerable interest in the isolation of new compounds from biological resources. The rapid emergence of fungal pathogens resistant to currently available antibiotics has further compounded the dearth of novel antifungal agents. The past decade has witnessed a dramatic growth in knowledge of natural peptides from plants, animals, and microorganisms. These peptides play an important role in the protection of plants from invasive infection and could prove to be useful tools for the genetic engineering of fungal resistance in transgenic plants (40).Antifungal peptides are classified into two classes based on their mode of action (17). The first group acts by lysis, which occurs via several mechanisms (34). Lytic peptides may be amphipathic, having two faces, with one being positively charged and the other being neutral and hydrophobic. The second class of peptide interferes with the cell wall synthesis or the biosynthesis of essential components (14). The biological activities of a large number of peptide toxins have been rationalized in terms of the peptides' having the ability to adopt amphiphilic ␣-helical structures (15,16,21). Peptides are expected to have value as alternative agents in the fight against new resistant microbial strains as they have modes of action different from those of classical antibiotics. The characterization of such new antifungal and antimicrobial peptides and the design of analogues with improved activities have allowed better ...

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Section: Resultsmentioning

confidence: 99%

Novel Bifunctional Inhibitor of Xylanase and Aspartic Protease: Implications for Inhibition of Fungal Growth

Dash

Ahmad

Nath

et al. 2001

Antimicrob Agents Chemother

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“…as described previously (23). Briefly, about 1000 ml of the extracellular culture filtrate was treated with 65 g of activated charcoal, and the supernatant was subjected to membrane filtration using Amicon UM10 (M r cut-off 10,000) and UM2 (M r cut-off 2000).…”

Section: Methodsmentioning

confidence: 99%

“…and was characterized for its inhibitory activity toward HIV-1 protease, pepsin, and the protease from Aspergillus saitoi (23,34,35). The bifunctional nature of ATBI was established by its potency toward Xyl I, the xylanase purified from the Thermomonospora sp.…”

Section: Kinetic Analysis Of the Inhibition Of Xyl I-mentioning

confidence: 99%

Slow-Tight Binding Inhibition of Xylanase by an Aspartic Protease Inhibitor

Dash

Vathipadiekal

George

et al. 2002

Journal of Biological Chemistry

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The first report of slow-tight inhibition of xylanase by a bifunctional inhibitor alkalo-thermophilic Bacillus inhibitor (ATBI), from an extremophilic Bacillus sp. is described. ATBI inhibits aspartic protease (Dash, C., and Rao, M. (2001) J. Biol. Chem., 276, 2487-2493) and xylanase (Xyl I) from a Thermomonospora sp. The steady-state kinetics revealed time-dependent competitive inhibition of Xyl I by ATBI, consistent with two-step inhibition mechanism. The inhibition followed a rapid equilibrium step to form a reversible enzyme-inhibitor complex (EI), which isomerizes to the second enzymeinhibitor complex (EI*), which dissociated at a very slow rate. The rate constants determined for the isomerization of EI to EI*, and the dissociation of EI* were 13 ؎ 1 ؋ 10 ؊6 s ؊1 and 5 ؎ 0.5 ؋ 10 ؊8 s ؊1 , respectively. The K i value for the formation of EI complex was 2.5 ؎ 0.5 M, whereas the overall inhibition constant K i * was 7 ؎ 1 nM. The conformational changes induced in Xyl I by ATBI were monitored by fluorescence spectroscopy and the rate constants derived were in agreement with the kinetic data. Thus, the conformational alterations were correlated to the isomerization of EI to EI*. ATBI binds to the active site of the enzyme and disturbs the native interaction between the histidine and lysine, as demonstrated by the abolished isoindole fluorescence of o-phthalaldehyde (OPTA)-labeled Xyl I. Our results revealed that the inactivation of Xyl I is due to the disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis and a model depicting the probable interaction between ATBI or OPTA with Xyl I has been proposed.

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“…However, the hydrophobic nature of pepstatin holds a disadvantage for its poor oral bioavailability. In our earlier report, we have shown that ATBI, 1 with the amino acid sequence of Ala-Gly-Lys-Lys-Asp-Asp-Asp-Asp-Pro-ProGlu, exhibited inhibitory activity toward HIV-1 protease (25). The present study deals with the evaluation of the kinetic parameters of ATBI, a slow-tight binding inhibitor of the aspartic proteases (AP), pepsin and F-prot from Aspergillus saitoi.…”

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confidence: 96%

Structural and Mechanistic Insight into the Inhibition of Aspartic Proteases by a Slow-Tight Binding Inhibitor from an ExtremophilicBacillussp.: Correlation of the Kinetic Parameters with the Inhibitor Induced Conformational Changes

et al. 2001

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We present here the first report of a hydrophilic peptidic inhibitor, ATBI, from an extremophilic Bacillus sp. exhibiting a two-step inhibition mechanism against the aspartic proteases, pepsin and F-prot from Aspergillus saitoi. Kinetic analysis shows that these proteases are competitively inhibited by ATBI. The progress curves are time-dependent and consistent with slow-tight binding inhibition: E + I right arrow over left arrow (k(3), k(4)) EI right arrow over left arrow (k(5), k(6)) EI. The K(i) values for the first reversible complex (EI) of ATBI with pepsin and F-prot were (17 +/- 0.5) x 10(-9) M and (3.2 +/- 0.6) x 10(-6) M, whereas the overall inhibition constant K(i) values were (55 +/- 0.5) x 10(-12) M and (5.2 +/- 0.6) x 10(-8) M, respectively. The rate constant k(5) revealed a faster isomerization of EI for F-prot [(2.3 +/- 0.4) x 10(-3) s(-1)] than pepsin [(7.7 +/- 0.3) x 10(-4) s(-1)]. However, ATBI dissociated from the tight enzyme-inhibitor complex (EI) of F-prot faster [(3.8 +/- 0.5) x 10(-5) s(-1)] than pepsin [(2.5 +/- 0.4) x 10(-6) s(-1)]. Comparative analysis of the kinetic parameters with pepstatin, the known inhibitor of pepsin, revealed a higher value of k(5)/k(6) for ATBI. The binding of the inhibitor with the aspartic proteases and the subsequent conformational changes induced were monitored by exploiting the intrinsic tryptophanyl fluorescence. The rate constants derived from the fluorescence data were in agreement with those obtained from the kinetic analysis; therefore, the induced conformational changes were correlated to the isomerization of EI to EI. Chemical modification of the Asp or Glu by WRK and Lys residues by TNBS abolished the antiproteolytic activity and revealed the involvement of two carboxyl groups and one amine group of ATBI in the enzymatic inactivation.

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Interactions of a Novel Inhibitor from an ExtremophilicBacillus sp. with HIV-1 Protease

Cited by 22 publications

References 40 publications

Novel Bifunctional Inhibitor of Xylanase and Aspartic Protease: Implications for Inhibition of Fungal Growth

Novel Bifunctional Inhibitor of Xylanase and Aspartic Protease: Implications for Inhibition of Fungal Growth

Slow-Tight Binding Inhibition of Xylanase by an Aspartic Protease Inhibitor

Structural and Mechanistic Insight into the Inhibition of Aspartic Proteases by a Slow-Tight Binding Inhibitor from an ExtremophilicBacillussp.: Correlation of the Kinetic Parameters with the Inhibitor Induced Conformational Changes

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