• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.140-144
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• 2003

Type Ⅰ signal peptidase is an integral membrane protein that functions to cleave signal peptides from secreted and membrane proteins. The enzyme serves as a potential target for the development of novel antibacterial agents due to its unique physiological properties. Despite being one of the best characterized enzymes, the catalysis of Type Ⅰ signal peptidase still remains controversy over the catalytic serine/lysine dyad mechanism. It appears that the dyad proteases are generally less efficient than the prototypical serine/histidine/aspartic acid triad found in most enzymes, although Type Ⅰ signal peptidase is an exception to this rule. In this paper, we have proposed that Type Ⅰ signal peptidase may act as the serine/lysine/aspartic acid triad cataltytic mechanism. Therefore, the aspartic acid 99 residue in the E. coli signal peptidase was chosen and mutated to an alanine to see if there is any possible role of the aspartic acid in the catalytic function. Type Ⅰ signal peptidase D99A protein was inactive in vitro assay using the procoat synthesized by in vitro transcription translation. However, the mutant was active using a highly sensitive in vivo assay. Pulse-chase experiments show that the replacement of aspartic acid 99 with alanine results in a very unstable signal peptidase molecule. Therefore, we conclude that it is unlikely that the residue is directly involved in catalysis, but rather plays an important role in stabilizing the protein structure.

• Molecules and Cells
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• v.39 no.8
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• pp.594-602
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• 2016

Alkyl hydroperoxide reductase subunit C from Pseudomonas aeruginosa PAO1 (PaAhpC) is a member of the 2-Cys peroxiredoxin family. Here, we examined the peroxidase and molecular chaperone functions of PaAhpC using a site-directed mutagenesis approach by substitution of Ser and Thr residues with Cys at positions 78 and 105 located between two catalytic cysteines. Substitution of Ser with Cys at position 78 enhanced the chaperone activity of the mutant (S78C-PaAhpC) by approximately 9-fold compared with that of the wild-type protein (WT-PaAhpC). This increased activity may have been associated with the proportionate increase in the high-molecular-weight (HMW) fraction and enhanced hydrophobicity of S78C-PaAhpC. Homology modeling revealed that mutation of $Ser^{78}$ to $Cys^{78}$ resulted in a more compact decameric structure than that observed in WT-PaAhpC and decreased the atomic distance between the two neighboring sulfur atoms of $Cys^{78}$ in the dimer-dimer interface of S78C-PaAhpC, which could be responsible for the enhanced hydrophobic interaction at the dimer-dimer interface. Furthermore, complementation assays showed that S78C-PaAhpC exhibited greatly improved the heat tolerance, resulting in enhanced1 survival under thermal stress. Thus, addition of Cys at position 78 in PaAhpC modulated the functional shifting of this protein from a peroxidase to a chaperone.

• Journal of Life Science
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• v.14 no.5
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• pp.752-760
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• 2004

The function of the [4Fe-4S] cluster containing iron (Fe-) protein in nitrogenase catalysis is to serve as the nucleotide-dependent electron donor to the MoFe protein which contains the sites for substrate binding and reduction. The ability of the Fe protein to function in this manner is dependent on its ability to adopt the appropriate conformation for productive interaction with the MoFe protein and on its ability to change redox potentials to provide the driving force required for electron transfer. The MgADP-bound (or off) conformational state of the nitrogenase Fe protein structure described reveals mechanisms for long-range communication from the nucleotide-binding sites to control affinity of association with the MoFe protein component. Two pathways, termed switches I and II, appear to be integral to this nucleotide signal transduction mechanism. In addition, the structure of the MgADP bound Fe protein provides the basis for the changes in the biophysical properties of the [4Fe-4S] observed when Fe protein binds nucleotides. The structures of the nitrogenase Fe protein with defined amino acid substitutions in the nucleotide dependent signal transduction pathways of the Switch I and Switch II have been determined by X-ray diffraction methods. These two pathways have been also implicated by site directed mutagenesis studies, structural analysis and analogies to other proteins that utilize similar nucleotide dependent signal transduction pathways. We have examined the validity of the assignment of these pathways in linking the signals generated by MgATP binding and hydrolysis to macromolecular complex formation and intermolecular electron transfer. The results provide a structural basis for the observed biophysical and biochemical properties of the Fe protein variants and interactions within the nitrogenase Fe protein-MoFe protein complex.

• BMB Reports
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• v.31 no.6
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• pp.595-599
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• 1998

To investigate conformational information of a low oxygen affinity recombinant hemoglobin (rHb) containing $96Val{\rightarrow}Trp$ mutation at the ${\alpha}96$ position, we ave produced rHb (${\alpha}96Val{\rightarrow}Phe$) and rHb (${\alpha}96Val{\rightarrow}Tyr$), using the Escherichia coli expression system and site-directed mutagenesis. The oxygen affinity of rHb (${\alpha}96Val{\rightarrow}Phe$) is similar to that of human normal adult hemoglobin (Hb A). However, the oxygen affinity of rHb (${\alpha}96Val{\rightarrow}Tyr$) showed much lower oxygen affinity than Hb A which is similar to that of rHb (${\alpha}96Val{\rightarrow}Tyr$), providing an opportunity as a potential candidate for a hemoglobin-based blood substitute. Both rHb (${\alpha}96Val{\rightarrow}Phe$) and rHb (${\alpha}96Val{\rightarrow}Tyr)$ showed high cooperativity in oxygen binding. IH-NMR spectroscopy shows that both rHb (${\alpha}96Val{\rightarrow}Phe$) and rHb (${\alpha}96Val{\rightarrow}Tyr$) have very similar tertiary structure around the heme pockets and uaternary structure in the ${\alpha}_1/{\beta}_2$ subunit interface ompared to Hb A. The low oxygen affinity of rHb (${\alpha}96Val{\rightarrow}Tyr$) has been suggested to be due to a hydrogen bond caused by an extra hydroxyl group not present in rHb (${\alpha}96Val{\rightarrow}Phe$). However, investigation of the carbonmonoxy form of rHb (${\alpha}96Val{\rightarrow}Phe$) and (${\alpha}96Val{\rightarrow}Try$) in the presence of inositol hexaphosphate at low temperature suggests that low oxygen affinity of (${\alpha}96Val{\rightarrow}Try$) may arise from a mechanism different to that of rHb (${\alpha}96Val{\rightarrow}Trp$).

• BMB Reports
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• v.37 no.6
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• pp.720-725
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• 2004

A number of signaling molecules contain small pleckstrin homology (PH) domains capable of binding phosphoinositides or proteins. Phospholipase C (PLC)-${\gamma}1$ has two putative PH domains, an $NH_2$-terminal (PH1) and a split PH domain ($nPH_2$ and $cPH_2$). We previously reported that the split PH domain of PLC-${\gamma}1$ binds to phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)$P_2$) (Chang et al., 2002). To identify the amino acid residues responsible for binding with PI(4)P and PI(4,5)$P_2$, we used site-directed mutagenesis to replace each amino acid in the variable loop-1 (VL-1) region of the PLC-${\gamma}1$ $nPH_2$ domain with alanine (a neutral amino acid). The phosphoinositide-binding affinity of these mutant molecules was analyzed by Dot-blot assay followed by ECL detection. We found that two PLC-${\gamma}1$ nPH2 domain mutants, P500A and H503A, showed reduced affinities for phosphoinositide binding. Furthermore, these mutant PLC-${\gamma}1$ molecules showed reduced PI(4,5)$P_2$ hydrolysis. Using green fluorescent protein (GFP) fusion protein system, we showed that both $PH_1$ and $nPH_2$ domains are responsible for membrane-targeted translocation of PLC-${\gamma}1$ upon serum stimulation. Together, our data reveal that the amino acid residues $Pro^{500}$ and $His^{503}$ are critical for binding of PLC-${\gamma}1$ to one of its substrates, PI(4,5)$P_2$ in the membrane.

• Journal of Life Science
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• v.6 no.3
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• pp.185-192
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• 1996

Bacteriophage T7 gene 2.5 protein, a single-stranded DNA binding protein, is required for T7 DNA replication, recombination, and repair. T7 gene 2.5 protein has two distinctive domains, DNA binding and C-terminal domain, directly involved in protein-protein interaction. Gene 2.5 protein participates in the DNA replication of Bacteriophage T7, which makes this protein essential for the T7 growth and DNA replication. What gene 2.5 protein makes important at T7 growth and DNA replication is its binding affinity to single-stranded DNA and the protein-protein important at T7 DNA replication proteins which are essential for the T7 DNA synthesis. We have constructed pGST2.5(WT) encoding the wild-type gene 2.5 protein and pGST2.5$\Delta $21C lacking C-terminal 21 amino acid residues. The purified GST-fusion proteins, GST2.5(WT) and GST2.5(WT)$\Delta$21C, were used for whether the carboxyl-terminal domain participates in the protein-protein interactions or not. GST2.5(WT) and GST2.5$\Delta$21C showed the difference in the protein-protein interaction. GST2.5(WT) interacted with T7 DNA polymerase and gene 4 protein, but GST2.5$\Delta$21C did not interact with either protein. Secondly, GST2.5(WT) interacts with gene 4 proteins (helicase/primase) but not GST2.5$\Delta$21C. these results proved the involvement of the carboxyl-terminal domain of gene 2.5 protein in the protein-protein interaction. We clearly conclude that carboxy-terminal domain of gene 2.5 protein is firmly involved in protein-protein interactions in T7 replication proteins.

• KSBB Journal
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• v.16 no.2
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• pp.188-199
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• 2001

A gene encoding the dextransucrase(dsCB) that synthesizes mostly $\alpha-(1\rightarrow6)$ linked dextran with low amount(10%) of $\alpha-(1\rightarrow3)$ branching was cloned and sequenced from Leuconostoc mesenteroides B-742CB. The 6.1 kbp DNA fragment carrying dsCB showed one open reading frame(ORF) composed of 4,536bp. The deduced amino acid sequence shows that it begins from the start codon(ATG) at position 698 of the cloned DNA fragment and extends to the termination condon(TAA) at position 5,223. The enzyme is consisted of 1,508 amino acids and has an calculated molecular mass of 168.6kDa. This calculated Mw was in good agreement with an activity band of 170kDa on non-denaturing SDS-PAGE. A recombinant E. coli DH5 $alpha$ harboring pDSCB produced extracellular dextransucrase in 2% sucrose medium, and synthesized both soluble and insoluble dextran. To compare the properties of enzyme with B-742CB dextransucrase, the acceptor reaction, hydrolysis of dextran and methylation were performed. The expressed enzyme showed the same properties as B-742CB dextransucrease, but its ability to synthesize $\alpha-(1\rightarrow3)$ branching was lower than that of B-742CB dextransucrase. In order to identify the critical amino acid residues known as conserved regions related to catalytic activity, Asp-492 was replaced with Asn. D492N resulted in a 1.6 fold decrease in specific activity.

• Molecules and Cells
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• v.41 no.12
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• pp.1061-1071
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• 2018

From Xenopus embryo studies, the BMP4/Smad1-targeted gene circuit is a key signaling pathway for specifying the cell fate between the ectoderm and neuro-ectoderm as well as the ventral and dorsal mesoderm. In this context, several BMP4/Smad1 target transcriptional factors have been identified as repressors of the neuro-ectoderm. However, none of these direct target transcription factors in this pathway, including GATA1b, Msx1 and Ventx1.1 have yet been proven as direct repressors of early neuro-ectodermal gene expression. In order to demonstrate that Ventx1.1 is a direct repressor of neuro-ectoderm genes, a genome-wide Xenopus ChIP-Seq of Ventx1.1 was performed. In this study, we demonstrated that Ventx1.1 bound to the Ventx1.1 response cis-acting element 1 and 2 (VRE1 and VRE2) on the promoter for zic3, which is a key early neuro-ectoderm gene, and this Ventx1.1 binding led to repression of zic3 transcription. Site-directed mutagenesis of VRE1 and VRE2 within zic3 promoter completely abolished the repression caused by Ventx1.1. In addition, we found both the positive and negative regulation of zic3 promoter activity by FoxD5b and Xcad2, respectively, and that these occur through the VREs and via modulation of Ventx1.1 levels. Taken together, the results demonstrate that the BMP4/Smad1 target gene, Ventx1.1, is a direct repressor of neuro-ectodermal gene zic3 during early Xenopus embryogenesis.

• Journal of Life Science
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• v.29 no.1
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• pp.123-128
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• 2019

The use of 16S rDNA is commonplace in the determination of prokaryotic species. However, it has limitations, and there are few studies at the genus level. We investigated conserved genes and metabolic pathways at the genus level in 28 strains of 13 genera of prokaryotes using the COG database (conserved genes) and MetaCyc database (metabolic pathways). Conserved genes compared to total genes (core genome) at the genus level ranged from 27.62%(Nostoc genus) to 71.76%(Spiribacter genus), with an average of 46.72%. The lower ratio of core genome meant the higher ratio of peculiar genes of a prokaryote, namely specific biological activities or the habitat may be varied. The ratio of common metabolic pathways at the genus level was higher than the ratio of core genomes, from 58.79% (Clostridium genus) to 96.31%(Mycoplasma genus), with an average of 75.86%. When compared among other genera, members of the same genus were positioned in the closest nodes to each other. Interestingly, Bacillus and Clostridium genera were positioned in closer nodes than those of the other genera. Archaebacterial genera were grouped together in the ortholog and metabolic pathway nodes in a phylogenetic tree. The genera Granulicella, Nostoc, and Bradyrhizobium of the Acidobacteria, Cyanobacteria, and Proteobacteria phyla, respectively, were grouped in an ortholog content tree. The results of this study can be used for (i) the identification of common genes and metabolic pathways at each phylogenetic level and (ii) the improvement of strains through horizontal gene transfer or site-directed mutagenesis.

• Journal of Microbiology and Biotechnology
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• v.29 no.3
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• pp.373-381
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• 2019

Site-directed mutagenesis was employed to generate five different triple point mutations in the double mutant (C295A/I86A) of Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) by computer-aided modeling with the aim of widening the small alkyl-binding pocket. TeSADH engineering enables the enzyme to accept sterically hindered substrates that could not be accepted by the wild-type enzyme. The underline in the mutations highlights the additional point mutation on the double mutant TeSADH introduced in this work. The catalytic efficiency ($k_{cat}/K_M$) of the ${\underline{M151A}}$/C295A/I86A triple TeSADH mutant for acetophenone increased about 4.8-fold higher than that of the double mutant. A 2.4-fold increase in conversion of 3'-methylacetophenone to (R)-1-(3-methylphenyl)-ethanol with a yield of 87% was obtained by using ${\underline{V115A}}$/C295A/I86A mutant in asymmetric reduction. The ${\underline{A85G}}$/C295A/I86A mutant also produced (R)-1-(3-methylphenyl)-ethanol (1.7-fold) from 3'-methylacetophenone and (R)-1-(3-methoxyphenyl)-ethanol (1.2-fold) from 3'-methoxyacetophenone, with improved yield. In terms of thermal stability, the ${\underline{M151A}}$/C295A/I86A and ${\underline{V115A}}$/C295A/I86A mutants significantly increased ${\Delta}T_{1/2}$ by $+6.8^{\circ}C$ and $+2.4^{\circ}C$, respectively, with thermal deactivation constant ($k_d$) close to the wild-type enzyme. The ${\underline{M151A}}$/C295A/I86A mutant reacts optimally at $70^{\circ}C$ with almost 4 times more residual activity than the wild type. Considering broad substrate tolerance and thermal stability together, it would be promising to produce (R)-1-(3-methylphenyl)-ethanol from 3'-methylacetophenone by ${\underline{V115A}}$/C295A/I86A, and (R)-1-phenylethanol from acetophenone by ${\underline{M151A}}$/C295A/I86A mutant, in large-scale bioreduction processes.