A surface-exposed GH26 ß-mannanase from Bacteroides ovatus: Structure, role, and phylogenetic analysis of BoMan26B.

The galactomannan utilization locus (BoManPUL) of the human gut bacterium Bacteroides ovatus encodes BoMan26B, a cell-surface-exposed endomannanase whose functional and structural features have been unclear. Our study now places BoMan26B in context with related enzymes and reveals the structural basis for its specificity. BoMan26B prefers longer substrates and is less restricted by galactose side-groups than the mannanase BoMan26A of the same locus. Using galactomannan, BoMan26B generated a mixture of (galactosyl) manno-oligosaccharides shorter than mannohexaose. Three defined manno-oligosaccharides had affinity for the SusD-like surface-exposed glycan-binding protein, predicted to be implicated in saccharide transport. Co-incubation of BoMan26B and the periplasmic α-galactosidase BoGal36A increased the rate of galactose release by about 10-fold compared with the rate without BoMan26B. The results suggested that BoMan26B performs the initial attack on galactomannan, generating oligosaccharides that after transport to the periplasm are processed by BoGal36A. A crystal structure of BoMan26B with galactosyl-mannotetraose bound in subsites -5 to -2 revealed an open and long active-site cleft with Trp-112 in subsite -5 concluded to be involved in mannosyl interaction. Moreover, Lys-149 in the -4 subsite interacted with the galactosyl side-group of the ligand. A phylogenetic tree consisting of GH26 enzymes revealed four strictly conserved GH26 residues and disclosed that BoMan26A and BoMan26B reside on two distinct phylogenetic branches (A and B). The three other branches contain lichenases, xylanases, or enzymes with unknown activities. Lys-149 is conserved in a narrow part of branch B, and Trp-112 is conserved in a wider group within branch B.

ß-Mannanase-catalyzed synthesis of alkyl mannooligosides.

ß-Mannanases catalyze the conversion and modification of ß-mannans and may, in addition to hydrolysis, also be capable of transglycosylation which can result in enzymatic synthesis of novel glycoconjugates. Using alcohols as glycosyl acceptors (alcoholysis), ß-mannanases can potentially be used to synthesize alkyl glycosides, biodegradable surfactants, from renewable ß-mannans. In this paper, we investigate the synthesis of alkyl mannooligosides using glycoside hydrolase family 5 ß-mannanases from the fungi Trichoderma reesei (TrMan5A and TrMan5A-R171K) and Aspergillus nidulans (AnMan5C). To evaluate ß-mannanase alcoholysis capacity, a novel mass spectrometry-based method was developed that allows for relative comparison of the formation of alcoholysis products using different enzymes or reaction conditions. Differences in alcoholysis capacity and potential secondary hydrolysis of alkyl mannooligosides were observed when comparing alcoholysis catalyzed by the three ß-mannanases using methanol or 1-hexanol as acceptor. Among the three ß-mannanases studied, TrMan5A was the most efficient in producing hexyl mannooligosides with 1-hexanol as acceptor. Hexyl mannooligosides were synthesized using TrMan5A and purified using high-performance liquid chromatography. The data suggests a high selectivity of TrMan5A for 1-hexanol as acceptor over water. The synthesized hexyl mannooligosides were structurally characterized using nuclear magnetic resonance, with results in agreement with their predicted ß-conformation. The surfactant properties of the synthesized hexyl mannooligosides were evaluated using tensiometry, showing that they have similar micelle-forming properties as commercially available hexyl glucosides. The present paper demonstrates the possibility of using ß-mannanases for alkyl glycoside synthesis and increases the potential utilization of renewable ß-mannans.

Structural and biochemical analyses of glycoside hydrolase families 5 and 26 ß-(1,4)-mannanases from Podospora anserina reveal differences upon manno-oligosaccharide catalysis.

The microbial deconstruction of the plant cell wall is a key biological process that is of increasing importance with the development of a sustainable biofuel industry. The glycoside hydrolase families GH5 (PaMan5A) and GH26 (PaMan26A) endo-ß-1,4-mannanases from the coprophilic ascomycete Podospora anserina contribute to the enzymatic degradation of lignocellulosic biomass. In this study, P. anserina mannanases were further subjected to detailed comparative analysis of their substrate specificities, active site organization, and transglycosylation capacity. Although PaMan5A displays a classical mode of action, PaMan26A revealed an atypical hydrolysis pattern with the release of mannotetraose and mannose from mannopentaose resulting from a predominant binding mode involving the -4 subsite. The crystal structures of PaMan5A and PaMan26A were solved at 1.4 and 2.85 Å resolution, respectively. Analysis of the PaMan26A structure supported strong interaction with substrate at the -4 subsite mediated by two aromatic residues Trp-244 and Trp-245. The PaMan26A structure appended to its family 35 carbohydrate binding module revealed a short and proline-rich rigid linker that anchored together the catalytic and the binding modules.

An Aspergillus nidulans ß-mannanase with high transglycosylation capacity revealed through comparative studies within glycosidase family 5.

ß-Mannanases are involved in the conversion and modification of mannan-based saccharides. Using a retaining mechanism, they can, in addition to hydrolysis, also potentially perform transglycosylation reactions, synthesizing new glyco-conjugates. Transglycosylation has been reported for ß-mannanases in GH5 and GH113. However, although they share the same fold and catalytic mechanism, there may be differences in the enzymes' ability to perform transglycosylation. Three GH5 ß-mannanases from Aspergillus nidulans, AnMan5A, AnMan5B and AnMan5C, which belong to subfamily GH5_7 were studied. Comparative studies, including the GH5_7 TrMan5A from Trichoderma reesei, showed some differences between the enzymes. All the enzymes could perform transglycosylation but AnMan5B stood out in generating comparably higher amounts of transglycosylation products when incubated with manno-oligosaccharides. In addition, AnMan5B did not use alcohols as acceptor, which was also different compared to the other three ß-mannanases. In order to map the preferred binding of manno-oligosaccharides, incubations were performed in H2 (18)O. AnMan5B in contrary to the other enzymes did not generate any (18)O-labelled products. This further supported the idea that AnMan5B potentially prefers to use saccharides as acceptor instead of water. A homology model of AnMan5B showed a non-conserved Trp located in subsite +2, not present in the other studied enzymes. Strong aglycone binding seems to be important for transglycosylation with saccharides. Depending on the application, it is important to select the right enzyme.

Expression and characterization of a Bifidobacterium adolescentis beta-mannanase carrying mannan-binding and cell association motifs.

The gene encoding ß-mannanase (EC 3.2.1.78) BaMan26A from the bacterium Bifidobacterium adolescentis (living in the human gut) was cloned and the gene product characterized. The enzyme was found to be modular and to contain a putative signal peptide. It possesses a catalytic module of the glycoside hydrolase family 26, a predicted immunoglobulin-like module, and two putative carbohydrate-binding modules (CBMs) of family 23. The enzyme is likely cell attached either by the sortase mechanism (LPXTG motif) or via a C-terminal transmembrane helix. The gene was expressed in Escherichia coli without the native signal peptide or the cell anchor. Two variants were made: one containing all four modules, designated BaMan26A-101K, and one truncated before the CBMs, designated BaMan26A-53K. BaMan26A-101K, which contains the CBMs, showed an affinity to carob galactomannan having a dissociation constant of 0.34 µM (8.8 mg/liter), whereas BaMan26A-53K did not bind, showing that at least one of the putative CBMs of family 23 is mannan binding. For BaMan26A-53K, k(cat) was determined to be 444 s(-1) and K(m) 21.3 g/liter using carob galactomannan as the substrate at the optimal pH of 5.3. Both of the enzyme variants hydrolyzed konjac glucomannan, as well as carob and guar gum galactomannans to a mixture of oligosaccharides. The dominant product from ivory nut mannan was found to be mannotriose. Mannobiose and mannotetraose were produced to a lesser extent, as shown by high-performance anion-exchange chromatography. Mannobiose was not hydrolyzed, and mannotriose was hydrolyzed at a significantly lower rate than the longer oligosaccharides.

Parental responses and catastrophizing in online cognitive behavioral therapy for pediatric functional abdominal pain: A mediation analysis of a randomized controlled trial.

Objective: To test if decreased parental protective behaviors, monitoring behaviors, and parental catastrophizing mediate relief of gastrointestinal symptoms in children 8-12 years with functional abdominal pain disorders (FAPDs). The study uses secondary data analyses of a randomized controlled trial in which exposure-based online cognitive behavioral therapy (ICBT) was found superior to treatment as usual in decreasing gastrointestinal symptoms. Methods: The ICBT included 10 weekly modules for children and 10 weekly modules for parents. Treatment as usual consisted of any medication, dietary adjustments, and healthcare visits that the participants engaged in during 10 weeks. All measures were self-assessed online by parents. Biweekly assessments of the Adult Responses to Children's Symptoms (ARCS), Protect and Monitor subscales, and the Pain Catastrophizing Scale, parental version (PCS-P) were included in univariate and multivariate growth models to test their mediating effect on the child's gastrointestinal symptoms assessed with the Pediatric Quality of Life Gastrointestinal Symptoms Scale (PedsQL). Results: A total of 90 dyads of children with FAPDs and their parents were included in the study, of which 46 were randomized to ICBT and 44 to treatment as usual. The PCS-P was found to mediate change in the PedsQL ab = 0.639 (95% CI 0.020-2.331), while the ARCS Monitor ab = 0.472 (95% CI -1.002 to 2.547), and Protect ab = -0.151 (95% CI -1.455 to 0.674) were not mediators of change. Conclusions: To target parental catastrophizing in ICBT for pediatric FAPDs is potentially important to reduce abdominal symptoms in children.

Rational engineering of mannosyl binding in the distal glycone subsites of Cellulomonas fimi endo-beta-1,4-mannanase: mannosyl binding promoted at subsite -2 and demoted at subsite -3.

To date, rational redesign of glycosidase active-site clefts has been mainly limited to the removal of essential functionalities rather than their introduction. The glycoside hydrolase family 26 endo-beta-1,4-mannanase from the soil bacterium Cellulomonas fimi depolymerizes various abundant plant mannans. On the basis of differences in the structures and hydrolytic action patterns of this wild-type (but recombinantly expressed) enzyme and a homologous mannanase from Cellvibrio japonicus, two nonconserved amino acid residues at two distal glycone-binding subsites of the C. fimi enzyme were substituted, Ala323Arg at subsite -2 and Phe325Ala at subsite -3, to achieve inverted mannosyl affinities in the respective subsites, mimicking the Ce. japonicus enzyme that has an Arg providing mannosyl interactions at subsite -2. The X-ray crystal structure of the C. fimi doubly substituted mannanase was determined to 2.35 A resolution and shows that the introduced Arg323 is in a position suitable for hydrogen bonding to mannosyl at subsite -2. We report steady-state enzyme kinetics and hydrolysis-product analyses using anion-exchange chromatography and a novel rapid mass spectrometric profiling method of (18)O-labeled products obtained using H(2)(18)O as a solvent. The results obtained with oligosaccharide substrates show that although the catalytic efficiency (k(cat)/K(m)) is wild-type-like for the engineered enzyme, it has an altered hydrolytic action pattern that stems from promotion of substrate binding at subsite -2 (due to the introduced Arg323) and demotion of it at subsite -3 (to which removal of Phe325 contributed). However, k(cat)/K(m) decreased approximately 1 order of magnitude with polymeric substrates, possibly caused by spatial repositioning of the substrate at subsite -3 and beyond for the engineered enzyme.

Advancing the HIV Pre-Exposure Prophylaxis Continuum: A Collaboration Between a Public Health Department and a Federally Qualified Health Center in the Southern United States.

Uptake of pre-exposure prophylaxis (PrEP) has been limited among black and Latino men who have sex with men (MSM), especially in the southern United States. Public health departments and federally qualified health centers (FQHCs) serving predominantly uninsured populations are uniquely positioned to improve access. We evaluated a novel PrEP collaboration between a public health department and an FQHC in North Carolina (NC). In May 2015, a PrEP program was initiated that included no-cost HIV/sexually transmitted infection screening at a public health department, followed by referral to a colocated FQHC for PrEP services. We profiled the PrEP continuum for patients entering the program until February 2018. PrEP initiators and noninitiators were compared using Wilcoxon rank-sum test for continuous variables and chi-square or Fisher's exact tests for categorical variables. Of 196 patients referred to the FQHC, 60% attended an initial appointment, 43% filled a prescription, 38% persisted in care for >3 months, and 30% reported >90% adherence at follow-up. Among those presenting for initial appointments (n = 117), most were MSM (n = 95, 81%) and black (n = 62, 53%); 21 (18%) were Latinx and 9 (8%) were trans persons. Almost half (n = 55) were uninsured. We found statistically significant differences between PrEP initiators versus noninitiators based on race/ethnicity (p = 0.02), insurance status (p = 0.05), and history of sex work (p = 0.05). In conclusion, this collaborative model of PrEP care was able to reach predominantly black and Latino MSM in the southern United States. Although sustainable, program strategies to improve steps along the PrEP care continuum are vital in this population.

Using technology to support HIV self-testing among MSM.

PURPOSE OF REVIEW: Technology-based HIV self-testing (HST) interventions have the potential to improve access to HIV testing among gay, bisexual, and other MSM, as well as address concerns about HST use, including challenges with linkage to appropriate follow-up services. This review examines studies that use technology-based platforms to increase or improve the experience of HST among MSM. RECENT FINDINGS: Seven published studies and eight funded studies were included in this review. Comprehensive prevention interventions with free HST kit distribution and interventions that provide free HST kits and support the HST process address a greater number of barriers (e.g., access, correct use of testing kits, and correct interpretation of results) than studies that only distribute free HST kits through technology-based platforms. SUMMARY: By addressing HIV-testing barriers and specific HST concerns, these interventions address a critical need to improve first time and repeat testing rates among MSM. Additional research is needed to determine the efficacy of recent formative HST interventions. If proven efficacious, scale-up of these strategies have the potential to increase HIV testing among MSM via expanded HST uptake.

Phylogenetic analysis and substrate specificity of GH2 ß-mannosidases from Aspergillus species.

Phylogenetic analysis of glycoside hydrolase family 2 including Aspergillus sequences and characterised ß-mannosidases from other organisms, clusters putative Aspergillus ß-mannosidases in two distinct clades (A and B). Aspergillus species have at least one paralog in each of the two clades. It appears that clade A members are extracellular and clade B members intracellular. Substrate specificity analysis of MndA of Aspergillus niger (clade A) and MndB of Aspergillus nidulans (clade B) show that MndB, in contrast to MndA, does not hydrolyse polymeric mannan and has probably evolved to hydrolyse small unbranched ß-mannosides like mannobiose. A 3D-model of MndB provides further insight.

The family II carbohydrate-binding module of xylanase CflXyn11A from Cellulomonas flavigena increases the synergy with cellulase TrCel7B from Trichoderma reesei during the hydrolysis of sugar cane bagasse.

Synergy between Cellulomonas flavigena xylanase CflXyn11A and Trichoderma reesei endoglucanase TrCel7B was assessed during hydrolysis of alkaline pretreated sugar cane bagasse (SCB) after 12-48 h, applying the individual enzymes and mixtures of the enzymes. A high degree of synergy (6.3) between CflXyn11A and TrCel7B in hydrolysis of SCB was observed after 12h in the equimolar mixture. A threefold decrease in the degree of synergy was observed with TrCel7B and the catalytic module of CflXyn11A; suggesting an important role played by the carbohydrate-binding module of CflXyn11A (CflXyn11A-CBM) in the observed synergy. Affinity electrophoresis and binding assays showed that CflXyn11A-CBM binds to xylans and to a lesser extent to cellulose. Our results suggest that synergy is more pronounced at early stages of hydrolysis. Furthermore, for the first time it is described that a CBM carried by a xylanase significantly enhances the synergy with a cellulase (threefold increase in synergy).