Exploration of Strategies for Mechanism-Based Inhibitor Design for Family GH99 endo-α-1,2-Mannanases

endo-α-1,2-Mannosidases and -mannanases, members of glycoside hydrolase family 99 (GH99), cleave α-Glc/Man-1,3-α-Man-OR structures within mammalian N-linked glycans and fungal α-mannan, respectively. They are proposed to act through a two-step mechanism involving a 1,2-anhydrosugar "epoxide" intermediate incorporating two conserved catalytic carboxylates. In the first step, one carboxylate acts as a general base to deprotonate the 2-hydroxy group adjacent to the fissile glycosidic bond, and the other provides general acid assistance to the departure of the aglycon. We report herein the synthesis of two inhibitors designed to interact with either the general base (α-mannosyl-1,3-(2-aminodeoxymannojirimycin), Man2NH2 DMJ) or the general acid (α-mannosyl-1,3-mannoimidazole, ManManIm). Modest affinities were observed for an endo-α-1,2-mannanase from Bacteroides thetaiotaomicron. Structural studies revealed that Man2NH2 DMJ binds like other iminosugar inhibitors, which suggests that the poor inhibition shown by this compound is not a result of a failure to achieve the expected interaction with the general base, but rather the reduction in basicity of the endocyclic nitrogen caused by introduction of a vicinal, protonated amine at C2. ManManIm binds with the imidazole headgroup distorted downwards, a result of an unfavourable interaction with a conserved active site tyrosine. This study has identified important limitations associated with mechanism-inspired inhibitor design for GH99 enzymes

Development of a Single-Sided Guarded Hot Plate Apparatus for Thermal Conductivity Measurements

This work presents the development of an experimental set-up for measurements of thermal conductivity of solid materials, such as ceramics, polymers, rubbers, glasses, biological materials, etc. whose thermal conductivity lies in the approximate range between 0.1 and 2 W/mk The set-up was designed on the principle of the single-sided guarded hot plate method. In order to find the optimal design for generation of traceable 1-D heat flux through an investigated 300 mm x 300 mm specimen, a numerical heat transfer finite element method analysis was performed. The principal components of the measuring apparatus, such as hot plate with thermopile, cold plate, and auxiliary hot plate, were constructed according to the obtained results of simulations. Software for the control of experiment and data acquisition was developed using the LabVIEW programming environment

A β-mannanase with a lysozyme-like fold and a novel molecular catalytic mechanism

The enzymatic cleavage of β-1,4-mannans is achieved by endo-β-1,4-mannanases, enzymes involved in germination of seeds and microbial hemicellulose degradation, and which have increasing industrial and consumer product applications. β- Mannanases occur in a range of families of the CAZy sequence-based glycoside hydrolase (GH) classification scheme including families 5, 26, and 113. In this work we reveal that β- mannanases of the newly described GH family 134 differ from other mannanase families in both their mechanism and tertiary structure. A representative GH family 134 endo-β-1,4-mannanase from a Streptomyces sp. displays a fold closely related to that of hen egg white lysozyme but acts with inversion of stereochemistry. A Michaelis complex with mannopentaose, and a product complex with mannotriose, reveal ligands with pyranose rings distorted in an unusual inverted chair conformation. Ab initio quantum mechanics/molecular mechanics metadynamics quantified the energetically accessible ring conformations and provided evidence in support of a 1C4 → 3H4 ‡ → 3S1 conformational itinerary along the reaction coordinate. This work, in concert with that on GH family 124 cellulases, reveals how the lysozyme fold can be co-opted to catalyze the hydrolysis of different polysaccharides in a mechanistically distinct manner

Relationship between optical transparency and nanostructural features of silica aerogels

Abstract Silica aerogels are considered to be of great promise for use in transparent thermal insulation systems in solar architecture. The optical transparency of these highly porous materials is influenced by the reaction parameters upon preparation and the precursor used. Previously it was shown that the specific extinction due to bulk scattering decreases both with increasing macroscopic density and increasing pH-value of the sol-gel starting solution. Recently, it was also found that within the measurement accuracy the light scattering intensity of the aerogel bulk equals the extrapolated small-angle X-ray scattering intensity towards scattering angle zero if both types of measurement are performed with respect to an absolute scale. In the meantime, ultra small-angle X-ray scattering measurements have been performed in order to close the gap in momentum space between light and conventional small-angle X-ray scattering. As a result it can be stated that the nearly isotropic (Rayleigh) scattering is caused by the same nanostructural inhomogeneities of the aerogel network which lead to the characteristic small-angle scattering pattern. As a consequence, the amount of isotropically scattered light and thus the optical extinction can be directly related to a quantity called the correlation volume. For a variety of silica aerogels, it is shown how the latter depends on the nanostructural features of the gel network, such as average particle size, interparticle arrangement, pore diameter and an ordering parameter, which accounts for concentration effects

Contribution of shape and charge to the inhibition of a family GH99 endo-α-1,2-mannanase

[Image: see text] 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 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-α-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 α-Man-1,3-noeuromycin, which is revealed to be the most potent inhibitor (K(D) 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

Using an in-vitro biofilm model to assess the virulence potential of Bacterial Vaginosis or non-Bacterial Vaginosis Gardnerella vaginalis isolates

Gardnerella vaginalis is the most common species found in bacterial vaginosis (BV). However, it is also present in a significant proportion of healthy women and G. vaginalis vaginal colonization does not always lead to BV. In an effort to better understand the differences between G. vaginalis isolated from women with a positive (BV) versus a negative (non-BV) diagnosis of BV, we compared the virulence potential of 7 BV and 7 non-BV G. vaginalis isolates and assessed the virulence factors related to biofilm formation, namely: initial adhesion and cytotoxic effect, biofilm accumulation, susceptibility to antibiotics, and transcript levels of the known vaginolysin, and sialidase genes. Furthermore, we also determined the ability of G. vaginalis to displace lactobacilli previously adhered to HeLa cells. Our results showed that non-BV strains were less virulent than BV strains, as suggested by the lower cytotoxicity and initial adhesion to Hela cells. Significant differences in expression of known virulence genes were also detected, further suggesting a higher virulence potential of the BV associated G. vaginalis. Importantly, we demonstrated that BV associated G. vaginalis were able to displace pre-coated vaginal protective lactobacilli and we hypothesize this to be a trigger for BV development.European Union funds (FEDER/COMPETE) and by national funds (FCT) under the project with reference FCOMP-01-0124-FEDER-008991 (PTDC/BIA-MIC/098228/2008). FCT Strategic Project of UID/BIO/04469/2013 unit the project NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte(ON.2 – O Novo Norte), QREN, FEDER, and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462). FCT individual fellowship SFRH/BD/93963/2013

Structural and mechanistic insights into a Bacteroides vulgatus retaining N-acetyl-β-galactosaminidase that uses neighbouring group participation

Bacteroides vulgatus is a member of the human microbiota whose abundance is increased in patients with Crohn's disease. We show that a B. vulgatus glycoside hydrolase from the carbohydrate active enzyme family GH123, BvGH123, is an N-acetyl-β-galactosaminidase that acts with retention of stereochemistry, and, through a 3-D structure in complex with Gal-thiazoline, provide evidence in support of a neighbouring group participation mechanism

Identifying metabolite markers for preterm birth in cervicovaginal fluid by magnetic resonance spectroscopy

Introduction Preterm birth (PTB) may be preceded by changes in the vaginal microflora and metabolite profiles. Objectives We sought to characterise the metabolite profile of cervicovaginal fluid (CVF) of pregnant women by 1H NMR spectroscopy, and assess their predictive value for PTB. Methods A pair of high-vaginal swabs was obtained from pregnant women with no evidence of clinical infection and grouped as follows: asymptomatic low risk (ALR) women with no previous history of PTB, assessed at 20–22 gestational weeks, g.w., n = 83; asymptomatic high risk (AHR) women with a previous history of PTB, assessed at both 20–22 g.w., n = 71, and 26–28 g.w., n = 58; and women presenting with symptoms of preterm labor (PTL) (SYM), assessed at 24–36 g.w., n = 65. Vaginal secretions were dissolved in phosphate buffered saline and scanned with a 9.4 T NMR spectrometer. Results Six metabolites (lactate, alanine, acetate, glutamine/glutamate, succinate and glucose) were analysed. In all study cohorts vaginal pH correlated with lactate integral (r = -0.62, p\0.0001). Lactate integrals were higher in the term ALR compared to the AHR (20–22 g.w.) women (p = 0.003). Acetate integrals were higher in the preterm versus term women for the AHR (20–22 g.w.) (p = 0.048) and SYM (p = 0.003) groups; and was predictive of PTB\37 g.w. (AUC 0.78; 95 % CI 0.61–0.95), and delivery within 2 weeks of the index assessment (AUC 0.84; 95 % CI 0.64–1) in the SYM women, whilst other metabolites were not. Conclusion High CVF acetate integral of women with symptoms of PTL appears predictive of preterm delivery, as well as delivery within 2 weeks of presentation

Structural and biochemical insights into the function and evolution of sulfoquinovosidases

An estimated 10 billion tonnes of sulfoquinovose (SQ) are produced and degraded each year. Prokaryotic sulfoglycolytic pathways catabolize sulfoquinovose (SQ) liberated from plant sulfolipid, or its delipidated form α-d-sulfoquinovosyl glycerol (SQGro), through the action of a sulfoquinovosidase (SQase), but little is known about the capacity of SQ glycosides to support growth. Structural studies of the first reported SQase (Escherichia coli YihQ) have identified three conserved residues that are essential for substrate recognition, but crossover mutations exploring active-site residues of predicted SQases from other organisms have yielded inactive mutants casting doubt on bioinformatic functional assignment. Here, we show that SQGro can support the growth of E. coli on par with d-glucose, and that the E. coli SQase prefers the naturally occurring diastereomer of SQGro. A predicted, but divergent, SQase from Agrobacterium tumefaciens proved to have highly specific activity toward SQ glycosides, and structural, mutagenic, and bioinformatic analyses revealed the molecular coevolution of catalytically important amino acid pairs directly involved in substrate recognition, as well as structurally important pairs distal to the active site. Understanding the defining features of SQases empowers bioinformatic approaches for mapping sulfur metabolism in diverse microbial communities and sheds light on this poorly understood arm of the biosulfur cycle