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Bioresource Technology Dec 20203-Hydroxyarginine (3-OH-Arg) is an important intermediate for the synthesis of viomycin, an important antibiotic for the clinical treatment of tuberculosis. An efficient...
3-Hydroxyarginine (3-OH-Arg) is an important intermediate for the synthesis of viomycin, an important antibiotic for the clinical treatment of tuberculosis. An efficient strategy for 3-OH-Arg production based on protein engineering and recombinant whole-cell biocatalysis was demonstrated for the first time. To avoid challenging product separation due to the generation of α-ketoglutarate (α-KG) in the system, the molar ratio of the substrates L-Arg and L-Glu was optimized to ensure the efficient production of 3-OH-Arg as well as the complete consumption of α-KG. Through the establishment of a fed-batch process, 3-OH-Arg and succinic acid (SA) production reached to 9.9 g/L and 5.98 g/L after 36 h of reaction under the optimized conditions. This is the highest biosynthetic yield of 3-OH-Arg achieved to date, potentially offering a promising strategy for commercial production of hydroxylated amino acids.
Topics: Biocatalysis; Ketoglutaric Acids; Protein Engineering; Succinic Acid
PubMed: 33099094
DOI: 10.1016/j.biortech.2020.124261 -
RNA (New York, N.Y.) Jan 2016Here we report that the specificity of peptide release in the ribosome on a nonstop mRNA by ArfA and RF2 is achieved by an induced-fit mechanism. Using RF2 that is...
Here we report that the specificity of peptide release in the ribosome on a nonstop mRNA by ArfA and RF2 is achieved by an induced-fit mechanism. Using RF2 that is methylated on the glutamine of its GGQ motif (RF2(m)), we show that methylation substantially increases the rate of ArfA/RF2-catalyzed peptide release on a nonstop mRNA that does not occupy the ribosomal A site, but has only a modest effect on k(cat) by the same proteins on longer nonstop mRNAs occupying the A site of the mRNA channel in the ribosome. Our data suggest that enhancement in the kcat of peptide release by ArfA and RF2 under the cognate decoding condition is the result of favorable conformational changes in the nonstop complex. We demonstrate a shared mechanism between canonical and nonstop termination, supported by similarities in the kinetic mechanisms in antibiotic inhibition and methylation-correlated enhancement in the rate of peptide release. Despite these similarities, our data suggest that nonstop termination differs from canonical pathway in the downstream event of recycling.
Topics: Biocatalysis; Escherichia coli; Escherichia coli Proteins; Methylation; Paromomycin; Peptide Chain Termination, Translational; Peptide Termination Factors; Peptides; Protein Biosynthesis; RNA-Binding Proteins; Ribosomes; Viomycin
PubMed: 26554029
DOI: 10.1261/rna.053082.115 -
Nature Communications Sep 2014The antibiotic streptomycin is widely used in the treatment of microbial infections. The primary mechanism of action is inhibition of translation by binding to the...
The antibiotic streptomycin is widely used in the treatment of microbial infections. The primary mechanism of action is inhibition of translation by binding to the ribosome, but how it enters the bacterial cell is unclear. Early in the study of this antibiotic, a mysterious streptomycin-induced potassium efflux preceding any decrease in viability was observed; it was speculated that this changed the electrochemical gradient such that streptomycin better accessed the cytoplasm. Here we use a high-throughput screen to search for compounds targeting the mechanosensitive channel of large conductance (MscL) and find dihydrostreptomycin among the 'hits'. Furthermore, we find that MscL is not only necessary for the previously described streptomycin-induced potassium efflux, but also directly increases MscL activity in electrophysiological studies. The data suggest that gating MscL is a novel mode of action of dihydrostreptomycin, and that MscL's large pore may provide a mechanism for cell entry.
Topics: Anti-Bacterial Agents; Dihydrostreptomycin Sulfate; Escherichia coli; Escherichia coli Proteins; High-Throughput Screening Assays; Ion Channels; Patch-Clamp Techniques; Potassium; Spectinomycin; Streptomycin; Viomycin
PubMed: 25205267
DOI: 10.1038/ncomms5891 -
Biophysical Chemistry Apr 2015The unique advantage of the single molecule approach is to reveal the inhomogeneous subpopulations in an ensemble. For example, smFRET (single molecule fluorescence...
The unique advantage of the single molecule approach is to reveal the inhomogeneous subpopulations in an ensemble. For example, smFRET (single molecule fluorescence resonance energy transfer) can identify multiple subpopulations based on the FRET efficiency histograms. However, identifying multiple FRET states with overlapping average values remains challenging. Here, we report a new concept and method to analyze the single molecule FRET data of a ribosome system. The main results are as follows: 1. based on a hierarchic concept, multiple ribosome subpopulations are identified. 2. The subpopulations are self-identified via the cross-correlation analysis of the FRET histogram profiles. The dynamic heterogeneity is tracked after 2 min intervals on the same ribosomes individually. 3. The major ribosome subpopulations exchange with each other with a certain pattern, indicating some correlations among the motions of the tRNAs and the ribosomal components. Experiments under the conditions of 20% glycerol or 1mM viomycin supported this conclusion.
Topics: Algorithms; Fluorescence Resonance Energy Transfer; RNA, Transfer; Ribosomes
PubMed: 25817384
DOI: 10.1016/j.bpc.2015.02.008