Mutational analysis to identify the residues essential for the acetyltransferase activity of GlmU in Bacillus subtilis

Wang, Meng; Huang, Minhua; Gu, Huawei; Li, Shan; Wang, Jufang

doi:10.1039/c7ra00086c

Cited by 3 publications

(3 citation statements)

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“…cell wall biosynthesis. 22 DGS also binds cysteine C38 of the ribulose-5-phosphate reductase TarJ (EC 50 = 33.4 mM, UniProt Code Q2G1B9), which is involved in the binding of the catalytic zinc ion. 21 TarJ is essential in the synthesis of poly(ribitol phosphate)teichoic acid, which are the main cell wall teichoic acids of S. aureus.…”

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confidence: 99%

Degrasyn exhibits antibiotic activity against multi-resistantStaphylococcus aureusby modifying several essential cysteines

Lee

Sieber

et al. 2020

Chem. Commun.

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confidence: 99%

Degrasyn exhibits antibiotic activity against multi-resistantStaphylococcus aureusby modifying several essential cysteines

Lee

Sieber

et al. 2020

Chem. Commun.

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“…The CeA peptide was purified with nickel ion affinity chromatography, as reported previously [ 39 ]. As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The samples were separated and analyzed with 16% tricine SDS-PAGE. The CeA fusion produced with the CF system was purified with nickel ion affinity chromatography, as previously reported [ 39 ].…”

Section: Methodsmentioning

confidence: 99%

Rapid and efficient production of cecropin A antibacterial peptide in Escherichia coli by fusion with a self-aggregating protein

et al. 2018

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BackgroundCecropin A (CeA), a natural cationic antimicrobial peptide, exerts potent antimicrobial activity against a broad spectrum of Gram-positive and Gram-negative bacteria, making it an attractive candidate substitute for antimicrobials. However, the low production rate and cumbersome, expensive processes required for both its recombinant and chemical synthesis have seriously hindered the exploitation and application of CeA. Here, we utilized a short β-structured self-aggregating protein, ELK16, as a fusion partner of CeA, which allowed the efficient production of high-purity CeA antibacterial peptide with a simple inexpensive process.ResultsIn this study, three different approaches to the production of CeA peptide were investigated: an affinity tag (His-tag)-fused protein expression system (AT-HIS system), a cell-free protein expression system (CF system), and a self-assembling peptide (ELK16)-fused protein expression system (SA-ELK16 system). In the AT-HIS and CF systems, the CeA peptide was obtained with purities of 92.1% and 90.4%, respectively, using one or more affinity-chromatographic purification steps. The procedures were tedious and costly, with CeA yields of only 0.41 and 0.93 μg/mg wet cell weight, respectively. Surprisingly, in the SA-ELK16 system, about 6.2 μg/mg wet cell weight of high-purity (approximately 99.8%) CeA peptide was obtained with a simple low-cost process including steps such as centrifugation and acetic acid treatment. An antimicrobial test showed that the high-purity CeA produced in this study had the same antimicrobial activity as synthetic CeA peptide.ConclusionsIn this study, we designed a suitable expression system (SA-ELK16 system) for the production of the antibacterial peptide CeA and compared it with two other protein expression systems. A high yield of high-purity CeA peptide was obtained with the SA-ELK16 system, which greatly reduced the cost and time required for downstream processing. This system may provide a platform for the laboratory scale production of the CeA antibacterial peptide.Electronic supplementary materialThe online version of this article (10.1186/s12896-018-0473-7) contains supplementary material, which is available to authorized users.

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Unique C-terminal extension and interactome of Mycobacterium tuberculosis GlmU impacts its in vivo function and the survival of the pathogen

Agarwal

Soni

Kumar

et al. 2021

Biochemical Journal

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N-acetyl glucosamine-1-phosphate uridyltransferase (GlmU) is a bifunctional enzyme involved in the biosynthesis of Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a critical precursor for the synthesis of peptidoglycan and other cell wall components. The absence of a homolog in eukaryotes makes GlmU an attractive target for therapeutic intervention. Mycobacterium tuberculosis GlmU (GlmUMt) has features, such as a C-terminal extension, that are not present in GlmU orthologs from other bacteria. Here, we set out to determine the uniqueness of GlmUMt by performing in vivo complementation experiments using RvDglmU mutant. We find that any deletion of the carboxy-terminal extension region of GlmUMt abolishes its ability to complement the function of GlmUMt. Results show orthologs of GlmU, including its closest ortholog, from Mycobacterium smegmatis, cannot complement the function of GlmUMt. Furthermore, the co-expression of GlmUMt domain deletion mutants with either acetyl or uridyltransferase activities failed to rescue the function. However, co-expression of GlmUMt point mutants with either acetyl or uridyltransferase activities successfully restored the biological function of GlmUMt, likely due to the formation of heterotrimers. Based on the interactome experiments, we speculate that GlmUMt participates in unique interactions essential for its in vivo function.

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Mutational analysis to identify the residues essential for the acetyltransferase activity of GlmU in Bacillus subtilis

Abstract: Amino acid mutation analysis and molecular modeling to verify the essential residues in acetyltransferase catalytic mechanism of Bs-GlmU.

Cited by 3 publications

References 44 publications

Degrasyn exhibits antibiotic activity against multi-resistantStaphylococcus aureusby modifying several essential cysteines

Degrasyn exhibits antibiotic activity against multi-resistantStaphylococcus aureusby modifying several essential cysteines

Rapid and efficient production of cecropin A antibacterial peptide in Escherichia coli by fusion with a self-aggregating protein

Unique C-terminal extension and interactome of Mycobacterium tuberculosis GlmU impacts its in vivo function and the survival of the pathogen

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