• 한국작물학회지
• /
• 제40권1호
• /
• pp.52-58
• /
• 1995

옥수수의 5-methyltryptophan 저 항성 돌연변이 주（MR1）로부터 anthranilate synthetase（AS）와 tryptophan synthetase（75）의 특성을 분석하였다. 대조구 식물로써 사용한 옥수수 품종 당진의 순계와 저항성주에 있어서 AS와 75의 효소 활성은 5-MT를 포함하지 않은 MS기본배지에서 생장시켰을 때는 차이를 보이지 않았다. 그러나, 25mg/L의 5-MT를 포함한 MS배지에서 생장한 MR1에 있어서 AS의 활성은 대조구보다 2배 높았다. 또한, 4mg/L의 L-tryptophan을 처리 했을때의 AS의 활성은 50％ 저해 되었다. MR1의 조추출물로부터 대조구와 동일한 활성저해율을 나타내기 위해서는 약 4배의 아미노산이 필요하였다. MR1의 75활성은 대조 식물보다 4배가 더 높았다. 그리 하여 tryptophan synthetase B subunit （TSB）를 encoding하는 유전자를 cloning하여 염기배열을 결정 하였다. TSB유전자는 상이한 기관으로부터 cloning된 TSB와 높은 상동성을 보였으며, 모든 발육단계에서 발현하였다. 띠orthern hybridization분석에서 MR1의 TSB발현량은 대조식물보다 높게 나타났다

• Journal of Microbiology and Biotechnology
• /
• 제11권5호
• /
• pp.798-803
• /
• 2001

Peptide synthetases produced from various microorganisms are multifunctional enzyme complexes and their substrates are recognized and activated by adenylation domains. To identify the substrate specificity of the peptide synthetase isolated from Bacillus subtilis 713, known to produce an antifungal peptide, two adenylation domains containing the minimal functional portion were expressed and purified. ATP-ppi exchange experiments and kinetic studies revealed that the two adenylation enzymes had a substrate specificity to $_{L}-lysine{\;}and{\;}_{L}-tryptophan$, respectively. In addition, based on a signature sequence comparison, the substrate of the third domain was predicted to be L-glutamic acid. These results suggest that this peptide synthetase is novel because there has been no previous report on a peptide synthetase that uses $_{L}-lysine,{\;}_{L}-tryptophan,{\;}and{\;}_{L}-glutamic$ acid as substrates in that order.

• 한국자기공명학회논문지
• /
• 제9권2호
• /
• pp.110-121
• /
• 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.

• 미생물학회지
• /
• 제30권6호
• /
• pp.564-569
• /
• 1992

녹황색 세균인 Chlorobium limicola f. thiosulfatophilum NCIB 8327을 glutamate 가 질소원으로 포함된 변형 Pfennig 배지에서 배양한 다음 균체를 얻고, glutamine synthetase 효소를 초원심분리, DEAE-Sepharose CL-6B 이온교환 크로마토그래피, Sephacryl S-300 젤여과 크로마토그래피, 분취용 액체크로마토그래피의 과정을 통해 2% 수율에 46.3배의 정제배수로효소를 분리정제하였다. UV-VIS 흡수스펙트럼과 polycrylamide 젤 정기영동을 통해분리된 효소의 순수도는 확인하였다. Sephacryl S-3000 젤을 이용하여 얻은 효소의 분자량은 280 kDa 정도였으며, SDS-polyacrylamide 젤 전기영동 결과, 30 kDa 정도의 분자랴ㄹ을 갖는 단위체의 심합체로 추정된다. 이효소의 최적 온도와 pH 는 각각 $30^{\circ}C$ 와 pH 7.0 이었으나, 두기질 L-glutamine 과 hydroxylamine-HCI 에 대한 km 값은 각각 27.9 mM 과 0.92 mM 이었다. 이 효소의 활성은 alamine, glycine, tryptophan 등의 아미노산에 의해 상당한 저해를 받는 것으로 나타났으며, asparagin, lysine, leucine, valine 등에 의해서는 거의 영향을 받지 않았다.

• 한국미생물·생명공학회지
• /
• 제14권5호
• /
• pp.427-432
• /
• 1986

The regulation of three ammonia assimilatory enzymes, GDH (glutamate dehydrogenase), GS (glutamine synthetase) and GOGAT (glutamate synthase), has been examined in C. glutamicum. Three kinds of arginine auxotrophs blocked in each step of arginine biosynthetic pathway from glutamate were selected as arg 5, arg 6, arg 8. Histidine and tryptophan auxotrophs were also selected because histidine and tryptophan repressed GS biosynthesis in E. coli. These strains were cultured on the media containing nitrogen-excess and limited conditions, to compare the specific activities of ${\alpha}$-ketoglutarate dehydrogenase(${\alpha}-KGDH$), GDH, GS, GOGAT from the cell-free extracts. These results showed that enzyme levels of ${\alpha}-KGDH$ and GDH from 3 kinds of arginine auxotrophs, histidine and tryptophan auxotrophs in nitrogen-excess condition and those of GS and GOGAT in nitrogen limited condition were increased compared with opposite condition. The tryptophan and histidine auxotrophs showed higher level of glutamate and glutamine than parental strains and other mutants. it is assumed that the higher levels of ${\alpha-KGDH}$ and GDH from mutants in nitrogen-excess condition promoted the accumulation of glutamate and glutamine in fermentation broth. The inhibition of GS activities by ADP suggested that GS is regulated by energy charge in C. glutamicum. The results with histidine, tryptophan, glycine, alanine, serine and GMP implied that a system of feedback inhibition were effective. The GDH, GS and GOGAT biosynthesis in culture broth was markedly repressed by the nature and kinds of available nitrogen sources such as tryptophan, proline, glycine, alanine, serine and tyrosine.

• BMB Reports
• /
• 제29권4호
• /
• pp.277-285
• /
• 1996

In Pseudomonas fluorescens grown on malonate as sole carbon source, acetyl-CoA synthetase was induced, suggesting that malonate is metabolized through acetate and then acetyl-CoA. Acetyl-CoA synthetase was purified 18.6-fold in 4 steps to apparent homogeneity. The native molecular mass of the enzyme estimated by a native acrylamide gel electrophoresis was 130 kDa. The enzyme was composed of two identical subunits with a molecular mass of 67 kDa. Optimum pH was 70. The acetyl-CoA synthetase showed typical Michaelis-Menten kinetics for the substrates, acetate, ATP and CoA, whose $K_m$ values were calculated to be 33.4, 74.8, and 40.7 mM respectively. Propionate. butyrate and pentanoate were also used as substrates by the enzyme, but the rate of the formation of the CoA derivatives was decreased in the order of the increase in carbon number. The enzyme was inhibited by the group-specific reagents diethylpyro-carbonate, 2,3-butanedione, pyridoxal-5'-phosphate and N-bromosuccinimide. In the presence of substrates the inactivation rate of the enzyme, by all of the group-specific reagents mentioned above decreased, indicating the presence of catalytically essential histidine, arginine, lysine and tryptophan residues at or near the active site. Preincubation of the enzyme with ATP, $Mg^{2+}$ resulted in the increase of its susceptibility to diethylpyrocarbonate, suggesting that ATP, $Mg^{2+}$ may induce a conformational change in the active site exposing the essential histidine residue to diethylpyrocarbonate. The enzyme was acetylated in the presence of acetyl-CoA, indicating that this is one of acyl-enzyme.

• 대한의생명과학회지
• /
• 제13권2호
• /
• pp.105-110
• /
• 2007

Aspartyl-tRNA synthetase (AspRS) exists in two different forms with respect to tRNA recognition. The discriminating enzyme (D-AspRS) recognizes only tRNA$^{Asp}$, while the non-discriminating one (ND-AspRS) also recognizes tRNA$^{Asn}$ and therefore forms both Asp-tRNA$^{Asn}$ and Asp-tRNA$^{Asp}$. Plus primary sequence distinguishes two general groups of AspRS. There is a predominantly bacterial-type, larger AspRS (about 580 aa) in addition to a shorter archaeal/eukaryotic type (about 430 aa). In vivo data made clear that discriminating and non-discriminating enzymes exist in both groups. The determinants in the protein sequence responsible for tRNA discrimination are not hewn. The AspRS from Acidithiobacillus ferrooxidans might be suggested ND-AspRS fur missing of AsnRS in genomic sequencing data. Therefore, we analyzed the AspRS from A. ferrooxidans with in vitro aminoacylation assay with E. coli unfractionated tRNA, in vivo missense suppression assay with tipA34 mutant and Northern hybridization with probes which were specific with tRNA$^{Asp}$ or tRNA$^{Asn}$. The AspRS from A. ferrooxidans produced more Asp-tRNA than that from E. coli. Only aspS gene from A. ferrooxidans suppressed trpA34 strain in minimal media without tryptophan. Only AspRS from A. ferrooxidans showed mischarged Asp-tRNA$^{Asn}$ band. Therefore, AspRS from A. ferrooxidans is definitely ND-AspRS.

• 한국미생물·생명공학회지
• /
• 제18권1호
• /
• pp.89-93
• /
• 1990

재조합 pBR322-trp$^+$ 플라스미드의 숙주내 안정적 유지를 목적으로 tRNA phe 의 구조유전자인 pheW$^+$ 유전자를 pBR322-trp$^+$의 플라스미드에 도입시키고, 숙주세포로는 트립토판 생산을 위한 정상숙주 LC901의 phenylalanyl tRNA synthetase 온도감수성 변이체인 LC901-pheS-ts를 구성하여 이 온도감수성 숙주의 제한온도 (restrictive temperature)에서 재조합 trp$^+$ 플라스미드의 안정적 유지와 trp$^+$ 유전자가 미치는 효과를 조사하였다.