Dataset Information

Discovery of a Kojibiose Phosphorylase in Escherichia coli K-12.

ABSTRACT: The substrate profiles for three uncharacterized enzymes (YcjM, YcjT, and YcjU) that are expressed from a cluster of 12 genes ( ycjM-W and ompG) of unknown function in Escherichia coli K-12 were determined. Through a comprehensive bioinformatic and steady-state kinetic analysis, the catalytic function of YcjT was determined to be kojibiose phosphorylase. In the presence of saturating phosphate and kojibiose (?-(1,2)-d-glucose-d-glucose), this enzyme catalyzes the formation of d-glucose and ?-d-glucose-1-phosphate ( kcat = 1.1 s-1, Km = 1.05 mM, and kcat/ Km = 1.12 × 103 M-1 s-1). Additionally, it was also shown that in the presence of ?-d-glucose-1-phosphate, YcjT can catalyze the formation of other disaccharides using 1,5-anhydro-d-glucitol, l-sorbose, d-sorbitol, or l-iditol as a substitute for d-glucose. Kojibiose is a component of cell wall lipoteichoic acids in Gram-positive bacteria and is of interest as a potential low-calorie sweetener and prebiotic. YcjU was determined to be a ?-phosphoglucomutase that catalyzes the isomerization of ?-d-glucose-1-phosphate ( kcat = 21 s-1, Km = 18 ?M, and kcat/ Km = 1.1 × 106 M-1 s-1) to d-glucose-6-phosphate. YcjU was also shown to exhibit catalytic activity with ?-d-allose-1-phosphate, ?-d-mannose-1-phosphate, and ?-d-galactose-1-phosphate. YcjM catalyzes the phosphorolysis of ?-(1,2)-d-glucose-d-glycerate with a kcat = 2.1 s-1, Km = 69 ?M, and kcat/ Km = 3.1 × 104 M-1 s-1.

SUBMITTER: Mukherjee K

PROVIDER: S-EPMC5953851 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Discovery of a Kojibiose Phosphorylase in Escherichia coli K-12.

Mukherjee Keya K Narindoshvili Tamari T Raushel Frank M FM

Biochemistry 20180430 19

The substrate profiles for three uncharacterized enzymes (YcjM, YcjT, and YcjU) that are expressed from a cluster of 12 genes ( ycjM-W and ompG) of unknown function in Escherichia coli K-12 were determined. Through a comprehensive bioinformatic and steady-state kinetic analysis, the catalytic function of YcjT was determined to be kojibiose phosphorylase. In the presence of saturating phosphate and kojibiose (α-(1,2)-d-glucose-d-glucose), this enzyme catalyzes the formation of d-glucose and β-d-g ...[more]

PMID: 29684280

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Project description:Despite the identification of many genes and pathways involved in the persistence phenomenon in bacteria, the mechanisms of persistence are not well understood. Here, using Escherichia coli, we identified polynucleotide phosphorylase (PNPase) as a key regulator of persister formation. We constructed the pnp knockout strain (Δpnp) and its complemented strain and exposed them to antibiotics and stress conditions. The results showed that, compared with the wild-type strain W3110, the Δpnp strain had significant defects in persistence to antibiotics and stresses, and the persistence phenotype was restored upon complementation with the pnp gene. Transcriptome sequencing (RNA-seq) analysis revealed that 242 (166 upregulated and 76 downregulated) genes were differentially expressed in the Δpnp strain compared with the W3110 strain. KEGG analysis of the upregulated genes showed that these genes were mostly mapped to metabolism and virulence pathways, of which most are positively regulated by the global regulator cyclic AMP receptor protein (CRP). Correspondingly, the transcription level of the crp gene in the Δpnp strain increased 3.22-fold in the early stationary phase. We further explored the indicators of cellular metabolism of the Δpnp strain, the phenotype of the pnp and crp double-deletion mutant, and the transcriptional activity of the crp gene. Our results indicate that PNPase controls cellular metabolism by negatively regulating the crp operon via targeting the 5'-untranslated region of the crp transcript. This study reveals a persister mechanism and provides novel targets for the development of drugs against persisters for more effective treatment. IMPORTANCE Persisters pose significant challenges for a more effective treatment of persistent infections. An improved understanding of mechanisms of persistence will provide therapeutic targets important for the development of better treatments. Since recent studies with the key tuberculosis persister drug pyrazinamide have implicated polynucleotide phosphorylase (PNPase) as a drug target, in this study, we addressed the possibility that PNPase might be involved in persistence in Escherichia coli. Our study demonstrates PNPase indeed being involved in persistence, provides a mechanism by which PNPase controls persister formation, and suggests a new therapeutic target for treating persistent bacterial infections.

Dataset Information

Discovery of a Kojibiose Phosphorylase in Escherichia coli K-12.

Publications

Discovery of a Kojibiose Phosphorylase in Escherichia coli K-12.

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