[A facile method for producing selenocysteine-containing proteins].
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ABSTRACT:
SUBMITTER: Mukai T
PROVIDER: S-EPMC6039127 | biostudies-literature | 2018 Jun
REPOSITORIES: biostudies-literature
Publications
[A facile method for producing selenocysteine-containing proteins].
Mukai Takahito T Sevostyanova Anastasia A Suzuki Tateki T Fu Xian X Söll Dieter D
Angewandte Chemie (Weinheim an der Bergstrasse, Germany) 20180406 24
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Project description:Selenocysteine (Sec, U) confers new chemical properties on proteins. Improved tools are thus required that enable Sec insertion into any desired position of a protein. We report a facile method for synthesizing selenoproteins with multiple Sec residues by expanding the genetic code of Escherichia coli. We recently discovered allo-tRNAs, tRNA species with unusual structure, that are as efficient serine acceptors as E. coli tRNASer . Ser-allo-tRNA was converted into Sec-allo-tRNA by Aeromonas salmonicida selenocysteine synthase (SelA). Sec-allo-tRNA variants were able to read through five UAG codons in the fdhF mRNA coding for E. coli formate dehydrogenase H, and produced active FDHH with five Sec residues in E. coli. Engineering of the E. coli selenium metabolism along with mutational changes in allo-tRNA and SelA improved the yield and purity of recombinant human glutathione peroxidase 1 (to over 80 %). Thus, our allo-tRNAUTu system offers a new selenoprotein engineering platform.
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Project description:The sulfate-reducing bacterium Desulfococcus multivorans uses various aromatic compounds as sources of cell carbon and energy. In this work, we studied the initial steps in the aromatic metabolism of this strictly anaerobic model organism. An ATP-dependent benzoate coenzyme A (CoA) ligase (AMP plus PPi forming) composed of a single 59-kDa subunit was purified from extracts of cells grown on benzoate. Specific activity was highest with benzoate and some benzoate derivatives, whereas aliphatic carboxylic acids were virtually unconverted. The N-terminal amino acid sequence showed high similarities with benzoate CoA ligases from Thauera aromatica and Azoarcus evansii. When cultivated on benzoate, cells strictly required selenium and molybdenum, whereas growth on nonaromatic compounds, such as cyclohexanecarboxylate or lactate, did not depend on the presence of the two trace elements. The growth rate on benzoate was half maximal with 1 nM selenite present in the growth medium. In molybdenum- and/or selenium-depleted cultures, growth on benzoate could be induced by addition of the missing trace elements. In extracts of cells grown on benzoate in the presence of [75Se]selenite, three radioactively labeled proteins with molecular masses of approximately 100, 30, and 27 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The 100- and 30-kDa selenoproteins were 5- to 10-fold induced in cells grown on benzoate compared to cells grown on lactate. These results suggest that the dearomatization process in D. multivorans is not catalyzed by the ATP-dependent Fe-S enzyme benzoyl-CoA reductase as in facultative anaerobes but rather involves unknown molybdenum- and selenocysteine-containing proteins.
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Project description:Selenocysteine (Sec or U) is encoded by UGA, a stop codon reassigned by a Sec-specific elongation factor and a distinctive RNA structure. To discover possible code variations in extant organisms we analyzed 6.4 trillion base pairs of metagenomic sequences and 24 903 microbial genomes for tRNA(Sec) species. As expected, UGA is the predominant Sec codon in use. We also found tRNA(Sec) species that recognize the stop codons UAG and UAA, and ten sense codons. Selenoprotein synthesis programmed by UAG in Geodermatophilus and Blastococcus, and by the Cys codon UGU in Aeromonas salmonicida was confirmed by metabolic labeling with (75) Se or mass spectrometry. Other tRNA(Sec) species with different anticodons enabled E. coli to synthesize active formate dehydrogenase H, a selenoenzyme. This illustrates the ease by which the genetic code may evolve new coding schemes, possibly aiding organisms to adapt to changing environments, and show the genetic code is much more flexible than previously thought.
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Project description:Here we report a novel structure-based microbial screening method for vinyl compound discovery, DISCOVER (direct screening method based on coupling reactions for vinyl compound producers). Through a two-step screening procedure based on selective coupling reactions of terminal alkenes, the thiol-ene reaction (1st step of screening) and Mizoroki-Heck reaction, followed by iodine test (2nd step of screening), microbes producing vinyl compounds like itaconic acid (IA) can be isolated from soil samples. In the 1st step of screening, soil sources are plated on agar medium supplemented with an antimicrobial agent, α-thioglycerol (TG), and a radical initiator, VA-044 (VA). In the 2nd step of screening, vinyl compounds produced in the cultures are labelled with iodobenzene via the Mizoroki-Heck reaction, followed by an iodine test, leading to the detection and characterisation of labelled products. We evaluated the validity of DISCOVER using IA and its producer Aspergillus terreus. Experimental data supported our hypothesis that IA reacts with TG in the medium via the thiol-ene reaction and consequently, A. terreus rapidly forms colonies on the agar medium because of the loss of the antimicrobial activity of TG. Using DISCOVER, high throughput and selective isolation of A. terreus strains producing IA was possible from soils.
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