• YAKHAK HOEJI
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• v.45 no.3
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• pp.245-250
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• 2001

Aminoacyl tRNA synthetases of bacteria are known as potential targets for new anti-microbial agents. To isolate new inhibitors of bacterial methionyl-tRNA synthetases from natural sources, a new target-oriented screening system using whole cells which are over-expressing a target enzyme was developed. Approximately 8,000 culture broths of microorganisms from soils were tested by this screening system. Among them, ten culture broths was found to contain inhibitory activity against methionyl -tRNA synthetases of Escherichia coli. For the validation of the screening system, this new method was compared with in vitro enzymatic method. Seven out of 10 culture broths showed inhibitory activity against methionyl-tRNA synthetases of E. coli. This result showed that the new screening system was comparable to the enzyme assay. Thus we believe that our screening system as a new method can be applied for the screening of new antibiotics inhibiting bacterial methionyl-tRNA synthetases from natural products.

• BMB Reports
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• v.28 no.4
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• pp.363-367
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• 1995

E. coli methionyl-tRNA synthetase is one of the class I tRNA synthetases. The Tryptophane residue at the position 461 located in the C-terminal domain of the enzyme is a key amino acid for the interaction with the anticodon of $tRNA^{Met}$. W461 was replaced with other amino acids to determine the chemical requirement for the interaction with the anticodon of $tRNA^{Met}$. Saturation mutagenesis at the position 461 generated a total of 12 substitution mutants of methionyl-tRNA synthetase. All the mutants showed the same in vivo stability as the wild-type enzyme, suggesting that the amino acid substitutions did not cause severe conformational change of the protein The mutants containing tyrosine, phenylalanine, histidine and cysteine substitutions showed in vivo activity while all the other mutants did not. The comparison of the in vitro aminoacylation activities of these mutants showed that aromatic ring structure, Van der Waals volume and hydrogen bond potential of the amino acid residue at the position 461 are the major determinants for the interaction with the anticodon of $tRNA^{Met}$.

• Journal of the Korean Magnetic Resonance Society
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• v.9 no.2
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• pp.110-121
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• 2005

E.coli methionyl tRNA synthetase consist of 676 amino acids and plays a key role in initiation of protein synthesis. The native form of this enzyme is a homodimer, but the monomeric enzyme truncated approximately C-terminal 120 amino acids retains the full enzymatic activities. X-ray crystal structure of the active monomeric enzyme shows that it has two domains. The N-terminal domain is thought to be a binding site for acceptor stem of tRNA, ATP, and methionine. The C-terminal domain is mainly a-helical and makes an interaction with the anticodon of $tRNA^{Met}$. Especially it is suggested that the region of helix-loop-helix including the tryptophan residue at the position 461 may be the essential for the interaction with anticodon of $tRNA^{Met}$. In this work the structure and function of E. coli methionyl-tRNA synthetase was studied by spectroscopic method (NMR, CD, Fluorescence). The importance of tryptophan residue at the position 461 was investigated by fluorescence spectroscopy. Tryptophan 461 is expected to be an essential site for the interaction between E. coli methionyl-tRNA synthetase and E. coli $tRNA^{Met}$. Proton and heteonuclear 2-dimensional NMR spectroscopy were also used to elucidate the protein-tRNA interaction.

• BMB Reports
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• v.32 no.6
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• pp.547-553
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• 1999

Aminoacyl-tRNA synthetases are essential enzymes catalyzing the attachment of specific amino acids to cognate tRNAs. In the present work, the substrate analogue L-methionine hydroxamate was used to identify functional residues located in the active site of the E. coli methionyl-tRNA synthetase (MetRS). This compound inhibited bacteria, yeast, and human MetRS activities to a similar degree, suggesting a conserved active site structure and mechanism between MetRSs of different phylogenetic domains. Mutants of the E. coli MetRS resistant to methionine hydroxamate were also isolated. These mutants contained a substitution either at T10, Y15, or Y94. These residues are highly conserved among the different MetRSs and the mutants showed decreased aminoacylation activity, suggesting their functional and structural significances. The putative roles of these residues are discussed on a structural basis.

• Journal of Life Science
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• v.28 no.2
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• pp.170-175
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• 2018

Parkinson's disease (PD) is the most common movement disorder and the second most common neurodegenerative disease after Alzheimer's disease. Approximately 5~10% of PD patients are familial PD cases. Leucine-rich repeat kinase 2 (LRRK2) has been known to be a causal gene of PD when it is mutated. LRRK2 contains the functional kinase and GTPase domains as well as leucine-rich repeat (LRR) and WD40 domains that are known to play critical roles for protein-protein interaction, suggesting that LRRK2-interacting proteins are important regulators for PD pathogenesis. In an effort to identify proteins interacting with LRRK2, we carried out co-immunoprecipitation of LRRK2 antibody using extracts of NIH3T3 cells that express LRRK2 at a relatively high level. The mass spectrometry analysis of a precipitated band revealed that the co-precipitated band was methionyl-tRNA synthetase (MRS), an ancient enzyme that transfers methionin to its cognate tRNA. The interaction of MRS with LRRK2 was confirmed again by co-immunoprecipitation of endogenous proteins and GST pull-down assays. However, LRRK2 did not phosphorylate recombinant MRS protein in in vitro kinase assays, and over-expression of LRRK2 or MRS did not affect the stability of its partner protein. Our data indicate that LRRK2 interacts with but does not phosphorylate MRS, and the stability of each partner is not affected by the other.

• Molecules and Cells
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• v.43 no.3
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• pp.304-311
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• 2020

Methionyl-tRNA synthetase (MRS) is essential for translation. MRS mutants reduce global translation, which usually increases lifespan in various genetic models. However, we found that MRS inhibited Drosophila reduced lifespan despite of the reduced protein synthesis. Microarray analysis with MRS inhibited Drosophila revealed significant changes in inflammatory and immune response genes. Especially, the expression of anti-microbial peptides (AMPs) genes was reduced. When we measured the expression levels of AMP genes during aging, those were getting increased in the control flies but reduced in MRS inhibition flies age-dependently. Interestingly, in the germ-free condition, the maximum lifespan was increased in MRS inhibition flies compared with that of the conventional condition. These findings suggest that the lifespan of MRS inhibition flies is reduced due to the down-regulated AMPs expression in Drosophila.

• Journal of Life Science
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• v.25 no.9
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• pp.1007-1013
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• 2015

A COG (Cluster of Orthologous Groups of proteins) algorithm was applied to detect conserved genes in 711 prokaryotes. Only COG0080 (ribosomal protein L11) was common among all the 711 prokaryotes analyzed and 58 COGs were common in more than 700 prokaryotes. Nine COGs among 58, including COG0197 (endonuclease III) and COG0088 (ribosomal protein L4), were conserved in a form of one gene per one organism. COG0008 represented 1356 genes in 709 of the prokaryotes and this was the highest number of genes among 58 COGs. Twenty-two COGs were conserved in more than 708 prokaryotes. Of these, two were transcription related, four were tRNA synthetases, eight were large ribosomal subunits, seven were small ribosomal subunits, and one was translation elongation factor. Among 58 conserved COGs in more than 700 prokaryotes, 50 (86.2%) were translation related, and four (6.9%) were transcription related, pointing to the importance of protein-synthesis in prokaryotes. Among these 58 COGs, the most conserved COG was COG0060 (isoleucyl tRNA synthetase), and the least conserved was COG0143 (methionyl tRNA synthetase). Archaea and eubacteria were discriminated in the genomic analysis by the average distance and variation in distance of common COGs. The identification of these conserved genes could be useful in basic and applied research, such as antibiotic development and cancer therapeutics.

• Journal of Life Science
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• v.28 no.2
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• pp.223-228
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• 2018

Lactic acid bacteria were isolated from a variety of fermented seafoods and sea creatures from the East Sea Rim, Korea and were screened for ${\gamma}-amino$ butyric acid-producing (GABA) activity. Through a 16S rRNA sequence analysis, the bacteria of interest, which were GABA-positive on the thin-layer chromatography analysis, were recognized as three isolates of Lactobacillus (Lb.) brevis and one isolate of Lactococcus (Lc.) lactis. Lb. brevis FSFL0004 and FSFL0005 were isolated from fermented anglerfish and Lb. brevis FSFL0036 was derived from salted cutlass fish. The Lc. lactis strain FGL0007 was isolated from the gut of a brown sole flounder. According to HPLC analysis, the GABA contents produced by FSFL0004, FSFL0005, FSFL0036 and FGL0007 were equivalent to $10,754.37{\mu}g/ml$, $13,082.79{\mu}g/ml$, $12,290.19{\mu}g/ml$, and $45.07{\mu}g/ml$ respectively in 1% monosodium glutamate-supplemented methionyl-tRNA synthetase (MRS) broth. The four strains were inoculated in skim milk with 1% monosodium glutamate to commercialize the strains as starter cultures for GABA-enriched dairy products, and TLC results displayed the production of ${\gamma}-amino$ butyric acid by all four strains in the adaptation media. Lc. lactis FGL0007 demonstrated the greatest GABA production ($431.42{\mu}g/ml$) by HPLC analysis. The GABA production by lactic acid bacteria strains in the skim milk demonstrated in the present study may be helpful for the production of GABA-enriched dairy products.