• Biomolecules & Therapeutics
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• v.14 no.1
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• pp.30-35
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• 2006

Tissue-type plasminogen activator (t-PA) is a multidomain serine protease containing two kringle domains, TK1-2. Previously, Pichia-derived recombinant human TK1-2 has been reported as an angiogenesis inhibitor although t-PA plays an important role in endothelial and tumor cell invasion. In this work, in order to improve in vivo efficacy of TK1-2 through elimination of immune reactivity, we mutated wild type TK1-2 into non-glycosylated form (NE-TK1-2) and examined whether it retains anti-angiogenic activity. The plasmid expressing NE-TK1-2 was constructed by replacing $Asn^{l17}\;and\;Asn^{184}$ with glutamic acid residues. After expression in Pichia pastoris, the secreted protein was purified from the culture broth using S-sepharose and UNO S1-FPLC column. The mass spectrum of NE-TK1-2 showed closely neighboring two peaks, 19631.87 and 19,835.44 Da, and it migrated as one band in SDS-PAGE. The patterns of CD-spectra of these two proteins were almost identical. Functionally, purified NE-TK1-2 was shown to inhibit endothelial cell migration in response to bFGF stimulation at the almost same level as wild type TK1-2. Therefore, the results suggest that non-glycosylated NETK1-2 can be developed as an effective anti-angiogenic and anti-tumor agent devoid of immune reactivity.

• BMB Reports
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• v.52 no.6
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• pp.403-408
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• 2019

Dorsoventral patterning of body axis in vertebrate embryo is tightly controlled by a complex regulatory network of transcription factors. Ventx1.1 is known as a transcriptional repressor to inhibit dorsal mesoderm formation and neural differentiation in Xenopus. In an attempt to identify, using chromatin immunoprecipitation (ChIP)-Seq, genome-wide binding pattern of Ventx1.1 in Xenopus gastrulae, we observed that Ventx1.1 associates with its own 5'-flanking sequence. In this study, we present evidence that Ventx1.1 binds a cis-acting Ventx1.1 response element (VRE) in its own promoter, leading to repression of its own transcription. Site-directed mutagenesis of the VRE in the Ventx1.1 promoter significantly abrogated this inhibitory autoregulation of Ventx1.1 transcription. Notably, Ventx1.1 and Xcad2, an activator of Ventx1.1 transcription, competitively co-occupied the VRE in the Ventx1.1 promoter. In support of this, mutation of the VRE down-regulated basal and Xcad2-induced levels of Ventx1.1 promoter activity. In addition, overexpression of Ventx1.1 prevented Xcad2 from binding to the Ventx1.1 promoter, and vice versa. Taken together, these results suggest that Ventx1.1 negatively regulates its own transcription in competition with Xcad2, thereby fine-tuning its own expression levels during dorsoventral patterning of Xenopus early embryo.

• The Plant Pathology Journal
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• v.37 no.6
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• pp.555-565
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• 2021

Bacteriophages infecting Acidovorax citrulli, the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King's B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.

• Bulletin of the Korean Chemical Society
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• v.31 no.9
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• pp.2497-2502
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• 2010

Arg13 is a conserved active-site residue in all known Pi class glutathione S-transferases (GSTs) and in most Alpha class GSTs. To evaluate its contribution to substrate binding and catalysis of this residue, three mutants (R13A, R13K, and R13L) were expressed in Escherichia coli and purified by GSH affinity chromatography. The substitutions of Arg13 significantly affected GSH-conjugation activity, while scarcely affecting glutathione peroxidase or steroid isomerase activities. Mutation of Arg13 into Ala largely reduced the GSH-conjugation activity by approximately 85 - 95%, whereas substitutions by Lys and Leu barely affected activity. These results suggest that, in the GSH-conjugation activity of hGST P1-1, the contribution of Arg13 toward catalytic activity is highly dependent on substrate specificities and the size of the side chain at position 13. From the kinetic parameters, introduction of larger side chains at position 13 results in stronger affinity (Leu > Lys, Arg > Ala) towards GSH. The substitutions of Arg13 with alanine and leucine significantly affected $k_{cat}$, whereas substitution with Lys was similar to that of the wild type, indicating the significance of a positively charged residue at position 13. From the plots of log ($k_{cat}/{K_m}^{CDNB}$) against pH, the $pK_a$ values of the thiol group of GSH bound in R13A, R13K, and R13L were estimated to be 1.8, 1.4, and 1.8 pK units higher than the $pK_a$ value of the wild-type enzyme, demonstrating the contribution of the Arg13 guanidinium group to the electrostatic field in the active site. From these results, we suggest that contribution of Arg13 in substrate binding is highly dependent on the nature of the electrophilic substrates, while in the catalytic mechanism, it stabilizes the GSH thiolate through hydrogen bonding.

• 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.

• Applied Biological Chemistry
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• v.48 no.3
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• pp.189-200
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• 2005

Biological nitrogen fixation is an important process for academic and industrial aspects. This review will briefly compare industrial and biological nitrogen fixation and cover the characteristics of biological nitrogen fixation studied in Azotobacter vinelandii. Various organisms can carry out biological nitrogen fixation and recently the researches on the reaction mechanism were concentrated on the free-living microorganism, A. vinelandii. Nitrogen fixation, which transforms atmospheric $N_2$ into ammonia, is chemically a reduction reaction requiring electron donation. Nitrogenase, the biological nitrgen fixer, accepts electrons from biological electron donors, and transfers them to the active site, FeMo-cofactor, through $Fe_4S_4$ cluster in Fe protein and P-cluster in MoFe protein. The electron transport and the proton transport are very important processes in the nitrogenase catalysis to understand its reaction mechanism, and the interactions between FeMo-cofactor and nitrogen molecule are at the center of biological nitrogen fixation mechanism. Spectroscopic studies including protein X-ray crystallography, EPR and $M{\ddot{o}}ssbauer$, biochemical approaches including substrate and inhibitor interactions as well as site-directed mutation study, and chemical approach to synthesize the FeMo-cofactor model compounds were used for biological nitrogen fixation study. Recent research results from these area were presented, and finally, a new nitrogenase reaction mechanism will be proposed based on the various research results.

• 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 (${\alpha}96Val{\rightarrow}Trp$).

• 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.

• 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.