X-ray Structure, Bioinformatics Analysis, and Substrate Specificity of a 6-Phospho-ß-glucosidase Glycoside Hydrolase 1 Enzyme from Bacillus licheniformis.

In bacteria, mono- and disaccharides are phosphorylated during the uptake processes through the vastly spread transport system phosphoenolpyruvate-dependent phosphotransferase. As an initial step in the phosphorylated disaccharide metabolism pathway, 6-phospho-ß-glucosidases and 6-phospho-ß-galactosidases play a crucial role by releasing phosphorylated and nonphosphorylated monosaccharides. However, structural determinants for the specificity of these enzymes still need to be clarified. Here, an X-ray structure of a glycoside hydrolase family 1 enzyme from Bacillus licheniformis, hereafter known as BlBglH, was determined at 2.2 Å resolution, and its substrate specificity was investigated. The sequence of BlBglH was compared to the sequences of 58 other GH1 enzymes using sequence alignments, sequence identity calculations, phylogenetic analysis, and motif discovery. Through these various analyses, BlBglH was found to have sequence features characteristic of the 6-phospho-ß-glucosidase activity enzymes. Motif and structural observations highlighted the importance of loop L8 in 6-phospho-ß-glucosidase activity enzymes. To further affirm enzyme specificity, molecular docking and molecular dynamics simulations were performed using the crystallographic structure of BlBglH. Docking was carried out with a 6-phospho-ß-glucosidase enzyme activity positive and negative control ligand, followed by 400 ns of MD simulations. The positive and negative control ligands were PNP6Pglc and PNP6Pgal, respectively. PNP6Pglc maintained favorable interactions within the active site until the end of the MD simulation, while PNP6Pgal exhibited instability. The favorable binding of substrate stabilized the loops that surround the active site. Binding free energy calculations showed that the PNP6Pglc complex had a substantially lower binding energy compared to the PNP6Pgal complex. Altogether, the findings of this study suggest that BlBglH possesses 6-phospho-ß-glucosidase enzymatic activity and revealed sequence and structural differences between bacterial GH1 enzymes of various activities.

Analysis of the galactomannan binding ability of ß-mannosidases, BtMan2A and CmMan5A, regarding their activity and synergism with a ß-mannanase.

Both ß-mannanases and ß-mannosidases are required for mannan-backbone degradation into mannose. In this study, two ß-mannosidases of glycoside hydrolase (GH) families 2 (BtMan2A) and 5 (CmMan5A) were evaluated for their substrate specificities and galactomannan binding ability. BtMan2A preferred short manno-oligomers, while CmMan5A preferred longer ones; DP >2, and galactomannans. BtMan2A displayed irreversible galactomannan binding, which was pH-dependent, with higher binding observed at low pH, while CmMan5A had limited binding. Docking and molecular dynamics (MD) simulations showed that BtMan2A galactomannan binding was stronger under acidic conditions (-8.4 kcal/mol) than in a neutral environment (-7.6 kcal/mol), and the galactomannan ligand was more unstable under neutral conditions than acidic conditions. Qualitative surface plasmon resonance (SPR) experimentally confirmed the reduced binding capacity of BtMan2A at pH 7. Finally, synergistic ß-mannanase to ß-mannosidase (BtMan2A or CmMan5A) ratios required for maximal galactomannan hydrolysis were determined. All CcManA to CmMan5A combinations were synergistic (≈1.2-fold), while combinations of CcManA with BtMan2A (≈1.0-fold) yielded no hydrolysis improvement. In conclusion, the low specific activity of BtMan2A towards long and galactose-containing oligomers and its non-catalytic galactomannan binding ability led to no synergy with the mannanase, making GH2 mannosidases ineffective for use in cocktails for mannan degradation.

AMBER force field parameters for the Zn (II) ions of the tunneling-fold enzymes GTP cyclohydrolase I and 6-pyruvoyl tetrahydropterin synthase.

The folate biosynthesis pathway is an essential pathway for cell growth and survival. Folate derivatives serve as a source of the one-carbon units in several intracellular metabolic reactions. Rapidly dividing cells rely heavily on the availability of folate derivatives for their proliferation. As a result, drugs targeting this pathway have shown to be effective against tumor cells and pathogens, but drug resistance against the available antifolate drugs emerged quickly. Therefore, there is a need to develop new treatment strategies and identify alternative metabolic targets. The two de novo folate biosynthesis pathway enzymes, GTP cyclohydrolase I (GCH1) and 6-pyruvoyl tetrahydropterin synthase (PTPS), can provide an alternative strategy to overcome the drug resistance that emerged in the two primary targeted enzymes dihydrofolate reductase and dihydropteroate synthase. Both GCH1 and PTPS enzymes contain Zn2+ ions in their active sites, and to accurately study their dynamic behaviors using all-atom molecular dynamics (MD) simulations, appropriate parameters that can describe their metal sites should be developed and validated. In this study, force field parameters of the GCH1 and PTPS metal centers were generated using quantum mechanics (QM) calculations and then validated through MD simulations to ensure their accuracy in describing and maintaining the Zn2+ ion coordination environment. The derived force field parameters will provide accurate and reliable MD simulations involving these two enzymes for future in-silico identification of drug candidates against the GCH1 and PTPS enzymes. Communicated by Ramaswamy H. Sarma.

Anti-HIV-1 integrase potency of methylgallate from Alchornea cordifolia using in vitro and in silico approaches.

According to the 2018 report of the United Nations Programme on HIV/AIDS (UNAIDS), acquired immune deficiency syndrome (AIDS), a disease caused by the human immunodeficiency virus (HIV), remains a significant public health problem. The non-existence of a cure or effective vaccine for the disease and the associated emergence of resistant viral strains imply an urgent need for the discovery of novel anti-HIV drug candidates. The current study aimed to identify potential anti-retroviral compounds from Alchornea cordifolia. Bioactive compounds were identified using several chromatographic and spectroscopic techniques and subsequently evaluated for cytotoxicity and anti-HIV properties. Molecular modelling studies against HIV-1 integrase (HIV-1 IN) were performed to decipher the mode of action of methylgallate, the most potent compound (IC50 = 3.7 nM) and its analogues from ZINC database. Cytotoxicity assays showed that neither the isolated compounds nor the crude methanolic extract displayed cytotoxicity effects on the HeLa cell line. A strong correlation between the in vitro and in silico results was observed and important HIV-1 IN residues interacting with the different compounds were identified. These current results indicate that methylgallate is the main anti-HIV-1 compound in A. cordifolia stem bark, and could be a potential platform for the development of new HIV-1 IN inhibitors.

Bioinformatic characterization of type-specific sequence and structural features in auxiliary activity family 9 proteins.

BACKGROUND: Due to the impending depletion of fossil fuels, it has become important to identify alternative energy sources. The biofuel industry has proven to be a promising alternative. However, owing to the complex nature of plant biomass, hence the degradation, biofuel production remains a challenge. The copper-dependent Auxiliary Activity family 9 (AA9) proteins have been found to act synergistically with other cellulose-degrading enzymes resulting in an increased rate of cellulose breakdown. AA9 proteins are lytic polysaccharide monooxygenase (LPMO) enzymes, otherwise known as polysaccharide monooxygenases (PMOs). They are further classified as Type 1, 2 or 3 PMOs, depending on the different cleavage products formed. As AA9 proteins are known to exhibit low sequence conservation, the analysis of unique features of AA9 domains of these enzymes should provide insights for the better understanding of how different AA9 PMO types function. RESULTS: Bioinformatics approaches were used to identify features specific to the catalytic AA9 domains of each type of AA9 PMO. Sequence analysis showed the N terminus to be highly variable with type-specific inserts evident in this region. Phylogenetic analysis was performed to cluster AA9 domains based on their types. Motif analysis enabled the identification of sub-groups within each AA9 PMO type with the majority of these motifs occurring within the highly variable N terminus of AA9 domains. AA9 domain structures were manually docked to crystalline cellulose and used to analyze both the type-specific inserts and motifs at a structural level. The results indicated that these regions influence the AA9 domain active site topology and may contribute to the regioselectivity displayed by different AA9 PMO types. Physicochemical property analysis was performed and detected significant differences in aromaticity, isoelectric point and instability index between certain AA9 PMO types. CONCLUSIONS: In this study, a type-specific characterisation of AA9 domains was performed using various bioinformatics approaches. These highly variable proteins were found to have a greater degree of conservation within their respective types. Type-specific features were identified for AA9 domains, which could be observed at a sequence, structural and physicochemical level. This provides a basis under which to identify and group new AA9 LPMOs in future.