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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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

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Author(s):
Veldman, Wayde [1] ; Liberato, Marcelo Vizona [2] ; Almeida, Vitor M. [3] ; Souza, Valquiria P. [3] ; Frutuoso, Maira A. [3] ; Marana, Sandro R. [3] ; Moses, Vuyani [1] ; Bishop, Ozlem Tastan [1] ; Polikarpov, Igor [2]
Total Authors: 9
Affiliation:
[1] Rhodes Univ, Dept Biochem & Microbiol, Res Unit Bioinformat RUBi, ZA-6140 Grahamstown - South Africa
[2] Univ Sao Paulo, Sao Carlos Inst Phys, BR-13566590 Sao Carlos - Brazil
[3] Univ Sao Paulo, Inst Quim, Dept Bioquim, BR-05508000 Sao Paulo - Brazil
Total Affiliations: 3
Document type: Journal article
Source: JOURNAL OF CHEMICAL INFORMATION AND MODELING; v. 60, n. 12, p. 6392-6407, DEC 28 2020.
Web of Science Citations: 0
Abstract

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-beta-glucosidases and 6-phospho-beta-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 angstrom 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-beta-glucosidase activity enzymes. Motif and structural observations highlighted the importance of loop L8 in 6-phospho-beta-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-beta-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-beta-glucosidase enzymatic activity and revealed sequence and structural differences between bacterial GH1 enzymes of various activities. (AU)

FAPESP's process: 15/13684-0 - Structural and functional studies of enzymes that participate in complex carbohydrates synthesis and degradation
Grantee:Igor Polikarpov
Support Opportunities: Research Projects - Thematic Grants