• Proceedings of the Microbiological Society of Korea Conference
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• 2004.05a
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• pp.15-16
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• 2004

• Proceedings of the Korean Society of Applied Pharmacology
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• 2002.07a
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• pp.236-236
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• 2002

Pyruvate dehydrogenase phosphatase (PDP) is a mitochondrial protein serine/threonine phosphatase that catalyzes the dephosphorylation and concomitant reactivation of the pyruvate dehydrogenase componant of the pyruvate dehydrogenase complex (PDC). PDP consists of a Mg$\^$+2/ -dependent and Ca$\^$+2)-stimulated catalytic subunit (PDPc) of Mr 52,600 and a FAD-containing regulatory subunit (PDPr) of Mr 95.600. Catalytic subunit of pyruvate dehydrogenase phosphatase (PDPc) has been suggested to have three major functional domains such as dihydrolipoamide acetyltransferase(E$_2$)-binding domain, regulatory subunit of PDP(PDPr)-binding domain, and calcium-binding domain. In order to identify functional domains, recombinant catalytic subunit of pyruvate dehydrogenase phosphatase (rPDPc) was expressed in E. coli JM101 and purified to near homogeneity using the unique property of PDPc: PDPm binds to the inner lipoyl domain (L$_2$) of E$_2$ of pyruvate dehydrogenase complex (PDC) in the presence of Ca$\^$+2/, not under EGTA. PDPc was limited-proteolysed by trypsin, chymotrypsin, Arg-C, and elastase at pH7.0 and 30$^{\circ}C$ and N-terminal analysis of the fragment was done. Chymotrypsin, trypsin, and elastase made two major framents: N-terminal large fragment, approx. 50kD and C-terminal small fragment, approx. 0 kDa. Arg-C made three major fragments: N-terminal fragment, approx. 35 kD, and central fragment, approx. 15 kD, and C-terminal fragment, approx. 10 kD. This study strongly suggest that PDPc consists of three major functional domains. However, further study should be necessary to identify the functional role.

• Journal of Microbiology and Biotechnology
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• v.25 no.8
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• pp.1227-1233
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• 2015

Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis through binding to its specific receptors, which mainly occurs to VEGF receptor 2 (VEGFR-2), a kinase insert domain-containing receptor. Therefore, the disruption of VEGFR-2 signaling provides a promising therapeutic approach for the treatment of cancer by inhibiting abnormal or tumorinduced angiogenesis. To explore this potential, we expressed the catalytic domain of VEGFR-2 (VEGFR-2-CD) as a soluble active kinase in Escherichia coli. The recombinant protein was purified and the VEGFR-2-CD activity was investigated. The obtained VEGFR-2-CD showed autophosphorylation activity and phosphate transfer activity comparable to the commercial enzyme. Furthermore, the IC₅₀ value of known VEGFR-2 inhibitor was determined using the purified VEGFR-2-CD. These results indicated a possibility for functional and economical VEGFR-2-CD expression in E. coli to use for inhibitor screening.

• Animal cells and systems
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• v.1 no.1
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• pp.135-142
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• 1997

Random sequencing of expressed sequence tags in roots of Chinese cabbage led to isolation of a partial cDNA clone, BR77, which encoded a putative protein kinase. Using the BR77 cDNA as a probe, we isolated a full-length cDNA encoding the Brassica campestris protein kinase 1 (Bcpk1). The Bcpt1 cDNA contained one open reading frame encoding a polypeptide of 439 amino acids. The putative polypeptide consisted of a short N-terminal region and a protein kinase catalytic domain. The catalytic domain of Bcpkl showed a high homology to cAMP- and calcium- phospholipid-dependent subfamilies of serine/threonine protein kineses. Eleven major catalytic domains in protein kineses were well conserved in Bcpk1. However, Bcpk1 contained a unique nonhomologous intervening sequence between subdomains VII and VIII, which was not found in protein kineses of animals and lower eukaryotes. Genomic DNA gel blot analysis showed that Bcpt1 genes might be present as three copies in the Chinese cabbage genome. These imply that Bcpk1 belongs to a plant-specific serine/threonine protein kinase subfamily.

• The Plant Pathology Journal
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• v.17 no.6
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• pp.365.1-365
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• 2001

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1453-1458
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• 2006

PKR-like endoplasmic reticulum (ER) kinase (PERK) is a type I transmembrane ER-resident protein containing a cytoplasmic catalytic domain with a Ser/Thr kinase activity, which is most closely related to the eukaryotic translation initiation factor-$2{\alpha}$ ($eIF2{\alpha}$) kinase PKR involved in the antiviral defense pathway by interferon. We cloned and expressed the PERK C-terminal kinase domain (cPERK) in Escherichia coli. Like PERK activation in cells under ER stress, wild-type cPERK underwent autophosphorylation when overexpressed in E. coli, whereas the cPERK(K621M) with a methionine substitution for the lysine at amino acid 621 lost the autophosphorylation activity. The activated form cPERK which was purified to near homogeneity, formed an oligomer and was able to trans-phosphorylate specifically its cellular substrate $eIF2{\alpha}$. Two-dimensional phosphoamino acids analysis revealed that phosphorylation of cPERK occurs at the Ser and Thr residues. The functionally active recombinant cPERK, and its inactive mutant should be useful for the analysis of biochemical functions of PERK and for the determination of their three-dimensional structures.

• Journal of Microbiology and Biotechnology
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• v.2 no.1
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• pp.56-65
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• 1992

The complete nucleotide sequence of the Bacillus circulans F-2 RSDA gene, coding for raw starch digesting a-amylase (RSDA), has been determined. The RSDA structure gene consists of an open reading frame of 2508 bp. Six bp upstream of the translational start codon of the RSDA is a typical gram-positive Shine-Dalgarno sequence and the RSDA encodes a preprotein of 836 amino acids with an Mr of 96, 727. The gene was expressed from its own regulatory region in E. coli and two putative consensus promoter sequences were identified upstream of a ribosome binding site and an ATG start codon. Confirmation of the nucleotide sequence was obtained and the signal peptide cleavage site was identified by comparing the predicted amino acid sequence with that derived by N-terminal analysis of the purified RSDA. The deduced N-terminal region of the RSDA conforms to the general pattern for the signal peptides of secreted prokaryotic proteins. The complete amino acid sequence was deduced and homology with other enzymes was compared. The results suggested that the Thr-Ser-rich hinge region and the non-catalytic domain are necessary for efficient adsorption onto raw substrates, and the catalytic domain (60 kDa) is necessary for the hydrolysis of substrates, as suggested in previous studies (8, 9).

• Journal of Microbiology and Biotechnology
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• v.19 no.11
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• pp.1306-1318
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• 2009

The filamentous ascomycete Sclerotinia sclerotiorum is well known for its ability to produce a large variety of hydrolytic enzymes. Two $\alpha$-amylases ScAmy54 and ScAmy43 predicted to play an important role in starch degradation were showed to produce specific oligosaccharides essentially maltotriose that have a considerable commercial interest. Primary structure of the two enzymes was established by N-terminal sequencing, MALDI-TOF masse spectrometry and cDNA cloning. The two proteins have the same N-terminal catalytic domain and ScAmy43 derived from ScAmy54 by truncation of 96 amino acids at the carboxyl-terminal region. Data of genomic analysis suggested that the two enzymes originated from the same $\alpha$-amylase gene and that truncation of ScAmy54 to ScAmy43 occurred probably during S. sclerotiorum cultivation. The structural gene of Scamy54 consisted of 9 exons and 8 introns, containing a single 1,500-bp open reading frame encoding 499 amino acids including a signal peptide of 21 residues. ScAmy54 exhibited high amino acid homology with other liquefying fungal $\alpha$-amylases essentially in the four conserved regions and in the putative catalytic triad. A 3D structure model of ScAmy54 and ScAmy43 was built using the 3-D structure of 2guy from A. niger as template. ScAmy54 is composed by three domains A, B, and C, including the well-known $(\beta/\alpha)_8$ barrel motif in domain A, have a typical structure of $\alpha$-amylase family, whereas ScAmy43 contained only tow domains A and B is the first fungal $\alpha$-amylase described until now with the smallest catalytic domain.

• KSBB Journal
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• v.19 no.1
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• pp.42-49
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• 2004

A gene coding for epoxide hydrolase (EH) of Aspergillus niger LK, a fungus possessing the enantioselective hydrolysis activity for racemic epoxides, was characterized by phylogenetic analysis. The deduced protein of A. niger LK epoxide hydrolase shares significant sequence similarity with several bacterial EHs and mammalian microsomal EHs (mEH) and belongs to the a/${\beta}$ hydrolase fold family. EH from A. niger LK had 90.6% identity with 3D crystal structure of lqo7 in Protein Data Bank. Sequence comparison with other source EHs suggested that Asp$\^$l92/, Asp$\^$374/ and His$\^$374/ constituted the catalytic triad. Based on the multiple sequence comparison of the functional and structural domain sequence, the phylogenetic tree between relevant epoxide hydrolases from various species were reconstructed by using Neighbor-Joining method. Genetic distances were so far as 1.841-2.682 but characteristic oxyanion hole and catalytic triad were highly conserved, which means they have diverged from a common ancestor.

• Journal of Life Science
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• v.21 no.3
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• pp.473-479
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• 2011

Adenylyl cyclase (AC) catalyzes the formation of cyclic AMP (cAMP) from ATP. The cAMP produced by AC serves as a secondary messenger in a variety of signal transduction pathways, and controls various cellular functions in many organisms. ACs can be grouped into six classes based on their primary amino acid sequences. Eukaryotes and mycobacteria contain only members of class III AC. The catalytic cyclase domains of class III AC are active as dimers: mammalian ACs, which are composed of a single polypeptide with two catalytic cyclase domains, form the active site as a result of intramolecular dimerization of the catalytic cyclase domains. In contrast, mycobacterial ACs function as homodimers, since their polypeptides contain a single catalytic cyclase domain. Six amino acids are required for the catalytic activity of class III AC - two aspartate residues, a lysine-aspartate pair and an arginine-asparagine pair. 16 ACs belonging to the class III were identified in Mycobacterium tuberculosis H37Rv, and their characteristics are reviewed.