A novel class of protein from wheat which inhibits xylanases1

We have purified a novel class of protein that can inhibit the activity of endo-β-1,4-xylanases. The inhibitor from wheat (Triticum aestivum, var. Soisson) is a glycosylated, monomeric, basic protein with a pI of 8.7–8.9, a molecular mass of 29 kDa and a unique N-terminal sequence of AGGKTGQVTVFWGRN. We have shown that the protein can inhibit the activity of two family-11 endo-β-1,4-xylanases, a recombinant enzyme from Aspergillus niger and an enzyme from Trichoderma viride.The inhibitory activity is heat and protease sensitive. The kinetics of the inhibition have been characterized with the A. niger enzyme using soluble wheat arabinoxylan as a substrate. The Km for soluble arabinoxylan in the absence of inhibitor is 20±2 mg/ml with a kcat of 103±6 s-1. The kinetics of the inhibition of this reaction are competitive, with a Ki value of 0.35 µM, showing that the inhibitor binds at or close to the active site of free xylanase. This report describes the first isolation of a xylanase inhibitor from any organism.

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TLXI, a novel type of xylanase inhibitor from wheat (Triticum aestivum) belonging to the thaumatin family

Biochemical Journal ◽

10.1042/bj20061291 ◽

2007 ◽

Vol 403 (3) ◽

pp. 583-591 ◽

Cited By ~ 91

Author(s):

Ellen Fierens ◽

Sigrid Rombouts ◽

Kurt Gebruers ◽

Hans Goesaert ◽

Kristof Brijs ◽

...

Keyword(s):

Triticum Aestivum ◽

Amino Acid ◽

Molecular Mass ◽

Glycoside Hydrolase ◽

Enzyme Inhibitor ◽

Glycoside Hydrolase Family ◽

Calculated Molecular Mass ◽

Sds Page ◽

Xylanase Inhibitor ◽

Hydrolase Family

Wheat (Triticum aestivum) contains a previously unknown type of xylanase (EC 3.2.1.8) inhibitor, which is described in the present paper for the first time. Based on its >60% similarity to TLPs (thaumatin-like proteins) and the fact that it contains the Prosite PS00316 thaumatin family signature, it is referred to as TLXI (thaumatin-like xylanase inhibitor). TLXI is a basic (pI≥9.3 in isoelectric focusing) protein with a molecular mass of approx. 18–kDa (determined by SDS/PAGE) and it occurs in wheat with varying extents of glycosylation. The TLXI gene sequence encodes a 26-amino-acid signal sequence followed by a 151-amino-acid mature protein with a calculated molecular mass of 15.6–kDa and pI of 8.38. The mature TLXI protein was expressed successfully in Pichia pastoris, resulting in a 21–kDa (determined by SDS/PAGE) recombinant protein (rTLXI). Polyclonal antibodies raised against TLXI purified from wheat react with epitopes of rTLXI as well as with those of thaumatin, demonstrating high structural similarity between these three proteins. TLXI has a unique inhibition specificity. It is a non-competitive inhibitor of a number of glycoside hydrolase family 11 xylanases, but it is inactive towards glycoside hydrolase family 10 xylanases. Progress curves show that TLXI is a slow tight-binding inhibitor, with a Ki of approx. 60–nM. Except for zeamatin, an α-amylase/trypsin inhibitor from maize (Zea mays), no other enzyme inhibitor is currently known among the TLPs. TLXI thus represents a novel type of inhibitor within this group of proteins.

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Characterization of an Abnormal Antithrombin (Milano 2) with Defective Thrombin Binding

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661681 ◽

1986 ◽

Vol 56 (03) ◽

pp. 349-352 ◽

Cited By ~ 2

Author(s):

A Tripodi ◽

A Krachmalnicoff ◽

P M Mannucci

Keyword(s):

Venous Thromboembolism ◽

Active Site ◽

Inhibitory Activity ◽

Antithrombin Iii ◽

Factor Xa ◽

Molecular Defect ◽

Affinity Adsorption ◽

Italian Family ◽

Crossed Immunoelectrophoresis

SummaryFour members of an Italian family (two with histories of venous thromboembolism) had a qualitative defect of antithrombin III reflected by normal antigen concentrations and halfnormal antithrombin activity with or without heparin. Anti-factor Xa activities were consistently borderline low (about 70% of normal). For the propositus’ plasma and serum the patterns of antithrombin III in crossed-immunoelectrophoresis with or without heparin were indistinguishable from those of normal plasma or serum. A normal affinity of antithrombin III for heparin was documented by heparin-sepharose chromatography. Affinity adsorption of the propositus’ plasma to human α-thrombin immobilized on sepharose beads revealed defective binding of the anti thrombin III to thrombin-sepharose. Hence the molecular defect of this variant appears to be at the active site responsible for binding and neutralizing thrombin, thus accounting for the low thrombin inhibitory activity.

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Characterization and kinetics of lipase inhibitory Activity of streptomyces tendae

International Journal of Pharma and Bio Sciences ◽

10.22376/ijpbs.2017.8.2.b270-274 ◽

2017 ◽

Vol 8 (2) ◽

Author(s):

GURMEET SINGH ◽

VENKATA KRISHNA BAYINENI ◽

RAVIKUMAR KADEPPAGARI

Keyword(s):

Inhibitory Activity ◽

Streptomyces Tendae ◽

Kinetics Of

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Trypsin entrapped within liposomes. Partition of a low-molecular-mass substrate as the main factor in kinetic control of hydrolysis

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19910712 ◽

1991 ◽

Vol 56 (3) ◽

pp. 712-717 ◽

Cited By ~ 2

Author(s):

Jana Formelová ◽

Albert Breier ◽

Peter Gemeiner ◽

Lubica Kurillová

Keyword(s):

Molecular Mass ◽

Egg Yolk ◽

Kinetic Control ◽

Hydrolysis Kinetics ◽

Liposomal Membrane ◽

Kinetics Of ◽

Main Factor

Trypsin has been entrapped within liposomes prepared from egg yolk phospholipides by the method of controlled dialysis, and the hydrolysis kinetics of Nα-benzoyl-DL-arginine p-nitroaniline catalyzed by the liposome-entrapped trypsin has been studied by monitoring the flux of substrate and product across the liposomal membrane. The partitioning of the substrate and product between liposomal and extraliposomal environment has been found to represent the main factor in the kinetic control of the hydrolysis.

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The melanocyte stimulating activity of N-nitroso-2-chloroethyl-carbamoyl derivatives of α-melanotropin fragments

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19882574 ◽

1988 ◽

Vol 53 (11) ◽

pp. 2574-2582 ◽

Cited By ~ 5

Author(s):

Hedvig Medzihradszky-Schweiger ◽

Helga Süli-Vargha ◽

József Bódi ◽

Kálmán Medzihradszky

Keyword(s):

Biological Activity ◽

Active Site ◽

Frog Skin ◽

Terminal Sequence ◽

Subsequent Reaction ◽

Affinity Labels ◽

Derivatives Of

A number of N-nitroso-2-chloroethyl-carbamoyl (Q(NO)) derivatives of α-melanotropin fragments have been synthesized and their effect on the frog skin melanocytes studied. Peptides substituted in this way possess the biological activity of the parent compounds, indicating that they preserved their receptor recognizing ability. These compounds can therefore serve as affinity labels. Some of these derivatives, related to the C-terminal sequence of α-melanotropin show prolonged darkening reaction, which does not influence the subsequent reaction of melanocytes with α-melanotropin. The Q(NO)-derivative of a fragment derived from the classical active site of the hormone shows, however, inhibition of the effect of α-melanotropin. It can be concluded that the latter peptide acts through the melanotropin receptor, while others, related to the C-terminal sequence of the hormone through another mechanism.

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Differences in Active Site Structure in a Family of β-glucan Endohydrolases Deduced from the Kinetics of Inactivation by Epoxyalkyl β-Oligoglucosides

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)83681-x ◽

1989 ◽

Vol 264 (9) ◽

pp. 4939-4947

Author(s):

P B Høj ◽

E B Rodriguez ◽

R V Stick ◽

B A Stone

Keyword(s):

Active Site ◽

Site Structure ◽

Active Site Structure ◽

Kinetics Of

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1250. Novel Boronic Acid Transition State Analogs (BATSI) with in vitro inhibitory activity against class A, B and C β-lactamases

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1434 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S643-S643

Author(s):

Maria F Mojica ◽

Christopher Bethel ◽

Emilia Caselli ◽

Magdalena A Taracila ◽

Fabio Prati ◽

...

Keyword(s):

Active Site ◽

Inhibitory Activity ◽

Covalent Bond ◽

Thiol Group ◽

Viable Cell ◽

Free Thiol ◽

Transition State Analogs ◽

Cell Counts ◽

Free Thiol Group

Abstract Background Catalytic mechanisms of serine β-lactamases (SBL; classes A, C and D) and metallo-β-lactamases (MBLs) have directed divergent strategies towards inhibitor design. SBL inhibitors act as high affinity substrates that -as in BATSIs- form a reversible, dative covalent bond with the conserved active site Ser. MBL inhibitors bind the active-site Zn2+ ions and displace the nucleophilic OH-. Herein, we explore the efficacy of a series of BATSI compounds with a free-thiol group at inhibiting both SBL and MBL. Methods Exploratory compounds were synthesized using stereoselective homologation of (+) pinandiol boronates to introduce the amino group on the boron-bearing carbon atom, which was subsequently acylated with mercaptopropanoic acid. Representative SBL (KPC-2, ADC-7, PDC-3 and OXA-23) and MBL (IMP-1, NDM-1 and VIM-2) were purified and used for the kinetic characterization of the BATSIs. In vitro activity was evaluated by a modified time-kill curve assay, using SBL and MBL-producing strains. Results Kinetic assays revealed that IC50 values ranged from 1.3 µM to >100 µM for this series. The best compound, s08033, demonstrated inhibitory activity against KPC-2, VIM-2, ADC-7 and PDC-3, with IC50 in the low μM range. Reduction of at least 1.5 log10-fold of viable cell counts upon exposure to sub-lethal concentrations of antibiotics (AB) + s08033, compared to the cells exposed to AB alone, demonstrated the microbiological activity of this novel compound against SBL- and MBL-producing E. coli (Table 1). Table 1 Conclusion Addition of a free-thiol group to the BATSI scaffold increases the range of these compounds resulting in a broad-spectrum inhibitor toward clinically important carbapenemases and cephalosporinases. Disclosures Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

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