• BMB Reports
• /
• 제42권1호
• /
• pp.47-52
• /
• 2009

Alanine racemase catalyzes the interconversion of L-alanine and D-alanine and plays a crucial role in spore germination and cell wall biosynthesis. In this study, alanine racemase produced by Bacillus anthracis was expressed and purified as a monomer in Escherichia coli and the importance of lysine 41 in the cofactor binding octapeptide and tyrosine 270 in catalysis was evaluated. The native enzyme exhibited an apparent $K_m$ of 3 mM for L-alanine, and a $V_{max}$ of $295\;{\mu}moles/min/mg$, with the optimum activity occurring at $37^{\circ}C$ and a pH of 8-9. The activity observed in the absence of exogenous pyridoxal 5'-phosphate suggested that the cofactor is bound to the enzyme. Additionally, the UV-visible absorption spectra indicated that the activity was pH independece, of VV-visible absorption spectra suggests that the bound PLP exists as a protonated Schiff's base. Furthermore, the loss of activity observed in the apoenzyme suggested that bound PLP is required for catalysis. Finally, the enzyme followed non-competitive and mixed inhibition kinetics for hydroxylamine and propionate with a $K_i$of $160\;{\mu}M$ and 30 mM, respectively.

• Asian Pacific Journal of Cancer Prevention
• /
• 제14권10호
• /
• pp.5789-5795
• /
• 2013

Background: The high mobility group box 1 (HMGB1) protein is a widespread nuclear protein present in most cell types. It typically locates in the nucleus and functions as a nuclear cofactor in transcription regulation. However, HMGB1 can also localize in the cytoplasm and be released into extracellular matrix, where it plays critical roles in carcinogenesis and inflammation. However, it remains elusive whether HMGB1 is relocated to cytoplasm in clear cell renal cell carcinoma (ccRCC). Methods: Nuclear and cytoplasmic proteins were extracted by different protocols from 20 ccRCC samples and corresponding adjacent renal tissues. Western blotting and immunohistochemistry were used to identify the expression of HMGB1 in ccRCC. To elucidate the potential mechanism of HMGB1 cytoplasmic translocation, HMGB1 proteins were enriched by immunoprecipitation and analyzed by mass spectrometry (MS). Results: The HMGB1 protein was overexpressed and partially localized in cytoplasm in ccRCC samples (12/20, 60%, p<0.05). Immunohistochemistry results indicated that ccRCC of high nuclear grade possess more HMGB1 relocation than those with low grade (p<0.05). Methylation of HMGB1 at lysine 112 in ccRCC was detected by MS. Bioinformatics analysis showed that post-translational modification might affect the binding ability to DNA and mediate its translocation. Conclusion: Relocation of HMGB1 to cytoplasm was confirmed in ccRCC. Methylation of HMGB1 at lysine 112 might the redistribution of this cofactor protein.

• 한국미생물·생명공학회지
• /
• 제18권1호
• /
• pp.49-55
• /
• 1990

D,L-2-aminothiazoline-4-carboxylic acid(D,L-ATC)로 부터 L-cysteine으로의 bioconversion에 대한 특성을 살펴보았다. Pseudomonas species의 배양중에 D,L-ATC를 첨가하여 균체내에 그 관여되는 효소를 유도, 생성시키고 균체만을 모은 후 파쇄하여 조효소액을 제조하였다. 실험결과, DL-ATC로 부터 L-형의 cysteine 만이 생성되며, 이 반응에 관여되는 효소는 cofactor로서 Mn이온을 필요로 하며, Mn 이온의 첨가에 의해 L-cysteine의 생성량이 수십배 증가되었다. 그러나, 이 효소는 생성물인 L-cysteine에 의해 feedback inhibition을 받았다. 한편, L-cysteine의 분해효소가 조효소액 내에 존재하며 그 효소반응의 저해제없이는 생성된 L-cysteine의 대부분이 분해되었다. 반면, 매우 효과적인 효소저해제인 hydroxylamine의 첨가로 L-cysteine의 분해를 거의 방지할 수 있었다.

• 한국미생물·생명공학회지
• /
• 제17권3호
• /
• pp.247-251
• /
• 1989

여러가지 염이 sisomicin 발효에 미치는 영향에 대해 살펴본 결과, CoCl$_2$만이 항생물질 생성의 대사반응에 cofactor로서 작용하였으며 16.8 $\mu$M에서 최대항생물질의 수율이 얻어졌다. 한편, 앞의 경우보다 훨씬 높은 염농도에서 ZnSO$_4$, KH$_2$PO$_4$, FeSO$_4$그리고 MgSO$_4$가 각각 발효배지에 첨가되었다. ZnSO$_4$와 KH$_2$PO$_4$는 전혀 효과가 없었으나 FeSO$_4$는 약간 항생물질 수율의 향상을 가져왔다. 그러나 MgSO$_4$의 경우, 매우 높은 염농도에서도 균체생육의 저해가 약간 일어났으며, 최종 항생물질의 수율은 100% 이상 증가되었다. 이러한 결과는 부분적으로 발효 중 균체내에 생성된 항생물질이 균체외로 유출되는 효과가 증진된 것에 기인한다.

• The Plant Pathology Journal
• /
• 제32권4호
• /
• pp.281-289
• /
• 2016

The transcription cofactor Swi6 plays important roles in regulating vegetative growth and meiosis in Saccharomyces cerevisiae. Functions of Swi6 ortholog were also characterized in Fusarium graminearum which is one of the devastating plant pathogenic fungi. Here, we report possible role of FgSwi6 in the interaction between F. graminearum and Fusarium graminearum virus 1 (FgV1) strain DK21. FgV1 perturbs biological characteristics of host fungi such as vegetative growth, sporulation, pigmentation, and reduction of the virulence (hypovirulence) of its fungal host. To characterize function(s) of FgSWI6 gene during FgV1 infection, targeted deletion, over-expression, and complementation mutants were generated and further infected successfully with FgV1. Deletion of FgSwi6 led to severe reduction of vegetative growth even aerial mycelia while over-expression did not affect any remarkable alteration of phenotype in virus-free isolates. Virus-infected (VI) FgSWI6 deletion isolate exhibited completely delayed vegetative growth. However, VI FgSWI6 over-expression mutant grew faster than any other VI isolates. To verify whether these different growth patterns in VI isolates, viral RNA quantification was carried out using qRT-PCR. Surprisingly, viral RNA accumulations in VI isolates were similar regardless of introduced mutations. These results provide evidence that FgSWI6 might play important role(s) in FgV1 induced phenotype alteration such as delayed vegetative growth.

• 한국자원식물학회지
• /
• 제19권6호
• /
• pp.706-715
• /
• 2006

Nitrate reductase deficient (NR) mutant lines were selected indirectly by their resistance to 100mM chlorate in cell cultures of P. parviflora. A total of 585 chlorate resistant lines were confirmed by a second passage on a high concentration of chlorate. Frequency of spontaneous mutation was $9.7{\times}10^{-7}$ in 3 month old suspension-cultured cells, and in non-selective media containing amino acids as sole nitrogen source. The frequency of mutation could be increased up to 11-fold by culture for 12 months. Out of 40 randomly selected calli, 22 were fully deficient in NR. The rest of the clones contained a decreased level of NR activity. Further characterization was carried out in 13 mutant lines which were fully deficient in NR and in 5 mutant lines containing residual (0-7.0%) NR activity, as compared to wild-type cells cultured on the same medium. The $NR^-$ mutants were tentatively classified as defective in the NR apoenzyme (nia-type; 11 mutant lines including the 5 with residual NR activity) or in the molybdenum cofactor (cnx-type; 7 mutant lines) by the XDH activity. The cnx-type could be further classified into two groups. In one group (5 mutant lines) of these, the NR activity could be partially restored by nonphysiologically high (1.0mM) molybdate in the culture medium. Both types of $NR^-$ mutants were unable to grow on minimal medium containing nitrate as sole nitrogen source, but grew well on amino acids. They also proved to be extremely sensitive to the standard medium ($MSP_1$) containing nitrate and ammonium. Shoot regeneration was obtained only in the $NR^-$ mutants, which contained residual NR activity, but they so far have failed to grow into plants.

• Archives of Pharmacal Research
• /
• 제19권6호
• /
• pp.480-485
• /
• 1996

Brain GABA transaminase is inactivated by preincubation with antidepressant/antipanic drug pheneizine (${\beta}$ethylphenylhydrazine) (mixing molar ratio 10:1) at pH 7.4. The reaction of enzyme with phenelzine was monitored by absorption and fluorescence spectroscopic methods. The inactive enzyme was fully reconstituted by addition of cofactor pyridoxal-5-phosphate. This result implies that the blocking of 1 mol of pyridoxal-5-phosphate per enzyme dimer is needed for inactivation of the enzyme. The time course of the reaction is significantly affected by the substrate .alpha.-ketoglutarate, which afforded complete protection against the loss of catalytic activity. The kinetic studies shows that phenelzine reacts with the cofactor of enzyme with a second-order rate constant of $2.1{\times}10^3M^{-1}s^{-1}$. It is postulated that the antidepressant/antipanic drug phenelzine is able to elevate the neurotransmitter GABA levels in central nervous system by inhibitory action on GABA degradative enzyme GABA transaminase.

• 한국미생물·생명공학회지
• /
• 제50권4호
• /
• pp.508-511
• /
• 2022

식물성 육류에서 미각적인 자극을 얻기 위해 사용하는 heme 함유 단백질의 지속적인 생산을 위해 강낭콩 유래 leghemoglobin a (PhLba) 유전자를 pPICZαA에 클로닝하고 Pichia pastoris에서 발현하였다. 재조합 PhLba 단백질은 가용화 형태로 배양 배지속으로 분비되었다. 정제된 PhLba의 분자량은 SDS-PAGE 상에서 16.5 kDa으로 나타났다. 재조합 PhLba holoprotein의 수율은 배양 배지에 hemin을 첨가함으로써 향상되었다. 이것은 PhLba의 apo 형태가 보조인자와 함께 효과적으로 포화된다는 것을 나타낸다.

• Journal of Microbiology
• /
• 제44권5호
• /
• pp.508-514
• /
• 2006

In this study, the double-stranded DNA-dependent activities of Deinococcus radiodurans RecA protein (Dr RecA) were characterized. The interactions of the Dr RecA protein with double-stranded DNA were determined, especially dsDNA-dependent ATP hydrolysis by the Dr RecA protein and the DNA strand exchange reaction, in which multiple branch points exist on a single RecA protein-DNA complex. A nucleotide cofactor (ATP or dATP ) was required for the Dr RecA protein binding to duplex DNA. In the presence of dATP, the nucleation step in the binding process occurred more rapidly than in the presence of ATP. Salts inhibited the binding of the Dr RecA protein to double-stranded DNA. Double-stranded DNA-dependent ATPase activities showed a different sensitivity to anion species. Glutamate had only a minimal effect on the double-stranded DNA-dependent ATPase activities, up to a concentration of 0.7 M. In the competition experiment for Dr RecA protein binding, the Dr RecA protein manifested a higher affinity to double-stranded DNA than was observed for single-stranded DNA.

• 한국결정학회지
• /
• 제16권2호
• /
• pp.66-74
• /
• 2005

다양한 생리활성을 가지는 marine natural products는 일반적인 secondary metabolites와 유사한 구조를 가지는데, 염소, 브롬, 요오드의 할로겐 원소에 의해 수식이 되어있는 것이 일반적이다. Vanadium haloperoxidase는 이러한 marine natural products의 생산에 중요한 효소로 vanadate를 조효소로 하는 금속효소이다. 본 리뷰에서는 vanadium haloperoxidase의 분리와 단백질 구조를 살펴보고, 이 금속효소의 작용기작에 대해서 설명할 것이다. 마지막으로, vanadium haloperoxidase의 반응성과 secondary metabolites 중 indole, terpenoids, acetogenins의 생합성 예를 살펴볼 것이다.