Dataset Information

Contribution of Shape and Charge to the Inhibition of a Family GH99 endo-?-1,2-Mannanase.

ABSTRACT: Inhibitor design incorporating features of the reaction coordinate and transition-state structure has emerged as a powerful approach for the development of enzyme inhibitors. Such inhibitors find use as mechanistic probes, chemical biology tools, and therapeutics. Endo-?-1,2-mannosidases and endo-?-1,2-mannanases, members of glycoside hydrolase family 99 (GH99), are interesting targets for inhibitor development as they play key roles in N-glycan maturation and microbiotal yeast mannan degradation, respectively. These enzymes are proposed to act via a 1,2-anhydrosugar "epoxide" mechanism that proceeds through an unusual conformational itinerary. Here, we explore how shape and charge contribute to binding of diverse inhibitors of these enzymes. We report the synthesis of neutral dideoxy, glucal and cyclohexenyl disaccharide inhibitors, their binding to GH99 endo-?-1,2-mannanases, and their structural analysis by X-ray crystallography. Quantum mechanical calculations of the free energy landscapes reveal how the neutral inhibitors provide shape but not charge mimicry of the proposed intermediate and transition state structures. Building upon the knowledge of shape and charge contributions to inhibition of family GH99 enzymes, we design and synthesize ?-Man-1,3-noeuromycin, which is revealed to be the most potent inhibitor (KD 13 nM for Bacteroides xylanisolvens GH99 enzyme) of these enzymes yet reported. This work reveals how shape and charge mimicry of transition state features can enable the rational design of potent inhibitors.

SUBMITTER: Petricevic M

PROVIDER: S-EPMC5269645 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Contribution of Shape and Charge to the Inhibition of a Family GH99 endo-α-1,2-Mannanase.

Petricevic Marija M Sobala Lukasz F LF Fernandes Pearl Z PZ Raich Lluís L Thompson Andrew J AJ Bernardo-Seisdedos Ganeko G Millet Oscar O Zhu Sha S Sollogoub Matthieu M Jiménez-Barbero Jesús J Rovira Carme C Davies Gideon J GJ Williams Spencer J SJ

Journal of the American Chemical Society 20170117 3

Inhibitor design incorporating features of the reaction coordinate and transition-state structure has emerged as a powerful approach for the development of enzyme inhibitors. Such inhibitors find use as mechanistic probes, chemical biology tools, and therapeutics. Endo-α-1,2-mannosidases and endo-α-1,2-mannanases, members of glycoside hydrolase family 99 (GH99), are interesting targets for inhibitor development as they play key roles in N-glycan maturation and microbiotal yeast mannan degradatio ...[more]

PMID: 27992199

Similar Datasets

Project description:BackgroundThe study of coffee polysaccharides-degrading enzymes from the coffee berry borer Hypothenemus hampei, has become an important alternative in the identification for enzymatic inhibitors that can be used as an alternative control of this dangerous insect. We report the cloning, expression and biochemical characterization of a mannanase gene that was identified in the midgut of the coffee berry borer and is responsible for the degradation of the most abundant polysaccharide in the coffee bean.MethodsThe amino acid sequence of HhMan was analyzed by multiple sequence alignment comparisons with BLAST (Basic Local Alignment Search Tool) and CLUSTALW. A Pichia pastoris expression system was used to express the recombinant form of the enzyme. The mannanase activity was quantified by the 3,5-dinitrosalicylic (DNS) and the hydrolitic properties were detected by TLC.ResultsAn endo-1,4-β-mannanase from the digestive tract of the insect Hypothenemus hampei was cloned and expressed as a recombinant protein in the Pichia pastoris system. This enzyme is 56% identical to the sequence of an endo-β-mannanase from Bacillus circulans that belongs to the glycosyl hydrolase 5 (GH5) family. The purified recombinant protein (rHhMan) exhibited a single band (35.5 kDa) by SDS-PAGE, and its activity was confirmed by zymography. rHhMan displays optimal activity levels at pH 5.5 and 30°C and can hydrolyze galactomannans of varying mannose:galactose ratios, suggesting that the enzymatic activity is independent of the presence of side chains such as galactose residues. The enzyme cannot hydrolyze manno-oligosaccharides such as mannobiose and mannotriose; however, it can degrade mannotetraose, likely through a transglycosylation reaction. The K(m) and k(cat) values of this enzyme on guar gum were 2.074 mg ml(-1) and 50.87 s(-1), respectively, which is similar to other mannanases.ConclusionThis work is the first study of an endo-1,4-β-mannanase from an insect using this expression system. Due to this enzyme's importance in the digestive processes of the coffee berry borer, this study may enable the design of inhibitors against endo-1,4-β-mannanase to decrease the economic losses stemming from this insect.

Dataset Information

Contribution of Shape and Charge to the Inhibition of a Family GH99 endo-?-1,2-Mannanase.

Publications

Contribution of Shape and Charge to the Inhibition of a Family GH99 endo-α-1,2-Mannanase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets