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Structural insights of two novel N-acetyl-glucosaminidase enzymes through in silico methods.

ABSTRACT: EndoBI-1 and EndoBI-2 are two endo- ?-N- acetylglucosaminidase isoenzymes that cleave N-N'- diacetylchitobiosyl moieties found in various types of native N -glycans. These N -glycans are indigestible by human infants and adults due to the lack of responsible glycosyl hydrolases and they act as selective prebiotics for a probiotic microorganism, Bifidobacterium longum subsp . infantis , in the large intestine. The selectivity and the thermostability of EndoBI-1 and EndoBI-2 suggest that these enzymes may be useful for many scientific and industrial applications. In this study, the growing numbers of homologous sequences in different databases were exploited in a comparative approach to investigate structural properties of EndoBI-1 and EndoBI-2 enzymes. Moreover, the complete and partial homology models of these two enzymes were generated and evaluated. Selected models were used for docking studies of the plus subsite ligand of these enzymes for further understanding on the substrate selectivity of EndoBI enzymes.

SUBMITTER: SahutoGlu AS

PROVIDER: S-EPMC7763110 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Structural insights of two novel N-acetyl-glucosaminidase enzymes through in silico methods.

ŞahutoĞlu Arif Sercan AS Duman Hatice H Frese Steven Alex SA Karav Sercan S

Turkish journal of chemistry 20201216 6

EndoBI-1 and EndoBI-2 are two endo- β-N- acetylglucosaminidase isoenzymes that cleave N-N'- diacetylchitobiosyl moieties found in various types of native N -glycans. These N -glycans are indigestible by human infants and adults due to the lack of responsible glycosyl hydrolases and they act as selective prebiotics for a probiotic microorganism, Bifidobacterium longum subsp . infantis , in the large intestine. The selectivity and the thermostability of En ...[more]

PMID: 33488263

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Project description:Background:N-Acetyl glucosamine (GlcNAc) and N-Acetyl chitooligosaccharides (N-Acetyl COSs) exhibit many biological activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Particularly, higher N-Acetyl COSs with degree of polymerization from 4 to 7 ((GlcNAc)4-(GlcNAc)7) show good antitumor and antimicrobial activity, as well as possessing strong stimulating activity toward natural killer cells. Thus, it is of great significance to discover a ?-N-acetyl glucosaminidase (NAGase) that can not only produce GlcNAc, but also synthesize N-Acetyl COSs. Results:The gene encoding the novel ?-N-acetyl glucosaminidase, designated CmNAGase, was cloned from Chitinolyticbacter meiyuanensis SYBC-H1. The deduced amino acid sequence of CmNAGase contains a glycoside hydrolase family 20 catalytic module that shows low identity (12-35%) with the corresponding domain of most well-characterized NAGases. The CmNAGase gene was highly expressed with an active form in Escherichia coli BL21 (DE3) cells. The specific activity of purified CmNAGase toward p-nitrophenyl-N-acetyl glucosaminide (pNP-GlcNAc) was 4878.6 U/mg of protein. CmNAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40 °C. The V max, K m, K cat, and K cat/K m of CmNAGase for pNP-GlcNAc were 16,666.67 ?mol min-1 mg-1, 0.50 ?mol mL-1, 25,555.56 s-1, and 51,111.12 mL ?mol-1 s-1, respectively. Analysis of the hydrolysis products of N-Acetyl COSs and colloidal chitin revealed that CmNAGase is a typical exo-acting NAGase. Particularly, CmNAGase can synthesize higher N-Acetyl COSs ((GlcNAc)3-(GlcNAc)7) from (GlcNAc)2-(GlcNAc)6, respectively, showed that it possesses transglycosylation activity. In addition, CmNAGase also has reverse hydrolysis activity toward GlcNAc, synthesizing various linked GlcNAc dimers. Conclusions:The observations recorded in this study that CmNAGase is a novel NAGase with exo-acting, transglycosylation, and reverse hydrolysis activities, suggest a possible application in the production of GlcNAc or higher N-Acetyl COSs.

Project description:The rise of antibiotic-resistant bacterial strains has become a critical health concern. According to the World Health Organization, the market introduction of new antibiotics is alarmingly sparse, underscoring the need for novel therapeutic targets. The LytR-CpsA-Psr (LCP) family of proteins, which facilitate the insertion of cell wall glycopolymers (CWGPs) like teichoic acids into peptidoglycan, has emerged as a promising target for antibiotic development. LCP proteins are crucial in bacterial adhesion and biofilm formation, making them attractive for disrupting these processes. This study investigated the structural and functional characteristics of the LCP domain of LytR from Streptococcus dysgalactiae subsp. dysgalactiae. The protein structure was solved by X-ray Crystallography at 2.80 Å resolution. Small-angle X-ray scattering (SAXS) data were collected to examine potential conformational differences between the free and ligand-bound forms of the LytR LCP domain. Additionally, docking and molecular dynamics (MD) simulations were used to predict the interactions and conversion of ATP to ADP and AMP. Experimental validation of these predictions was performed using malachite green activity assays. The determined structure of the LCP domain revealed a fold highly similar to those of homologous proteins while SAXS data indicated potential conformational differences between the ligand-free and ligand-bound forms, suggesting a more compact conformation during catalysis, upon ligand binding. Docking and MD simulations predicted that the LytR LCP domain could interact with ADP and ATP and catalyze their conversion to AMP. These predictions were experimentally validated by malachite green activity assays, confirming the protein's functional versatility. The study provides significant insights into the structural features and functional capabilities of the LCP domain of LytR from S. dysgalactiae subsp. dysgalactiae. These findings pave the way for designing targeted therapies against antibiotic-resistant bacteria and offer strategies to disrupt bacterial biofilm formation.

Dataset Information

Structural insights of two novel N-acetyl-glucosaminidase enzymes through in silico methods.

Publications

Structural insights of two novel N-acetyl-glucosaminidase enzymes through in silico methods.

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