• Plant Biotechnology Reports
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• v.5 no.4
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• pp.331-344
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• 2011

The well-conserved NBS domain of resistance (R) genes cloned from many plants allows the use of a PCR-based approach to isolate resistance gene analogs (RGAs). In this study, we isolated an RGA (CapRGC) from Capsicum annuum "CM334" using a PCR-based approach. This sequence encodes a protein with very high similarity to Rx genes, the Potato Virus X (PVX) R genes from potato. An evolutionary analysis of the CapRGC gene and its homologs retrieved by an extensive search of a Solanaceae database provided evidence that Rx-like genes (eight ESTs or genes that show very high similarity to Rx) appear to have diverged from R1 [an NBS-LRR R gene against late blight (Phytophthora infestans) from potato]-like genes. Structural comparison of the NBS domains of all the homologs in Solanaceae revealed that one novel motif, 14, is specific to the Rx-like genes, and also indicated that several other novel motifs are characteristic of the R1-like genes. Our results suggest that Rx-like genes are ancient but conserved. Furthermore, the novel conserved motifs can provide a basis for biochemical structural. function analysis and be used for degenerate primer design for the isolation of Rx-like sequences in other plant species. Comparative mapping study revealed that the position of CapRGC is syntenic to the locations of Rx and its homolog genes in the potato and tomato, but cosegregation analysis showed that CapRGC may not be the R gene against PVX in pepper. Our results confirm previous observations that the specificity of R genes is not conserved, while the structure and function of R genes are conserved. It appears that CapRGC may function as a resistance gene to another pathogen, such as the nematode to which the structure of CapRGC is most similar.

• Proceedings of the Korea Crystallographic Association Conference
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• 2002.11a
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• pp.4-4
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• 2002

PDZ domains bind to short segments within target proteins in a sequence-specific fashion. GRIP/ABP family proteins contain six to seven PDZ domains and interact via its sixth PDZ domain (class Ⅱ) with the C-termini of various proteins, including liprin-α. In addition the PDZ456 domain mediates the formation of homo- and heteromultimers of GRIP proteins. To better understand the structural basis of peptide recognition by a class Ⅱ PDZ domain and DZ-mediated multimerization, we determined the crystal structures of the GRIPI PDZ6 domain, alone and in complex with a synthetic C-terminal octapeptide of human liprin-α, at resolutions of 1.5 Å and 1.8 Å, respectively. Remarkably, unlike other class Ⅱ PDZ domains, Ile736 at αB5 rather than conserved Leu732 at αB1 makes a direct hydrophobic contact with the side chain of the Tyr at the -2 position of the ligand. Moreover, the peptide-bound structure of PDZ6 shows a slight reorientation of helix αB, indicating that the second hydrophobic pocket undergoes a conformational adaptation to accommodate the bulkiness of the Tyr's side chain, and forms an antiparallel dimer through an interface located at a site distal to the peptide-binding groove. This configuration may enable formation of GRIP multimers and efficient clustering of GRIP-binding proteins.

• Genomics & Informatics
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• v.21 no.3
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• pp.30.1-30.13
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• 2023

Ephs belong to the largest family of receptor tyrosine kinase and are highly conserved both sequentially and structurally. The structural organization of Eph is similar to other receptor tyrosine kinases; constituting the extracellular ligand binding domain, a fibronectin domain followed by intracellular juxtamembrane kinase, and SAM domain. Eph binds to respective ephrin ligand, through the ligand binding domain and forms a tetrameric complex to activate the kinase domain. Eph-ephrin regulates many downstream pathways that lead to physiological events such as cell migration, proliferation, and growth. Therefore, considering the importance of Eph-ephrin class of protein in tumorigenesis, 7,620 clinically reported missense mutations belonging to the class of variables of unknown significance were retrieved from cBioPortal and evaluated for pathogenicity. Thirty-two mutations predicted to be pathogenic using SIFT, Polyphen-2, PROVEAN, SNPs&GO, PMut, iSTABLE, and PremPS in-silico tools were found located either in critical functional regions or encompassing interactions at the binding interface of Eph-ephrin. However, seven were reported in nonsmall cell lung cancer (NSCLC). Considering the relevance of receptor tyrosine kinases and Eph in NSCLC, these seven mutations were assessed for change in the folding pattern using molecular dynamic simulation. Structural alterations, stability, flexibility, compactness, and solvent-exposed area was observed in EphA3 Trp790Cys, EphA7 Leu749Phe, EphB1 Gly685Cys, EphB4 Val748Ala, and Ephrin A2 Trp112Cys. Hence, it can be concluded that the evaluated mutations have potential to alter the folding pattern and thus can be further validated by in-vitro, structural and in-vivo studies for clinical management.

• Journal of Life Science
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• v.13 no.6
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• pp.879-888
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• 2003

In order to measure the enzyme activity of 5-epi-aristolochene hydroxylase, one of cytochrome P450 (P450) enzymes in eicitor-treated pepper cell, we used in vivo assay method and demonstrated a dramatic suppression of the activity by P450-inhibitors, ancymidol and ketocornazole. Using RT-PCR method with degenerate primer of the well conserved domains found within most P450-enzymes, and using cDNA library screening method, one distinct cDNA, being designated P450Hy01, was successfully isolated from elicitor-treated pepper cells. P450Hy01 mRNA was all induced in elicitor-treated cells whereas never induced in control cells. Moreover, levels of P450Hy01 expression were highly correlated with the levels of extracellular capsidiol production by different elicitors in cell cultures. P450Hy01 transcript was also induced by several other elicitors such as, cellulase, arachidonic acid, jasmonic acid, yeast extract as well as UV stress. P450Hy01 sequence contained high probability amino acid matches to known Plant P450 genes and ORF with a conserved FxxGxRxCxG heme-binding domain. P450Hy01 cDNA showed 98% of homology in sequence of nucleotide as well as amino acid to 5-epi-aristolochene-1, 3-hydroxylase (5EAl, 3H) which has been isolated in tobacco cells, suggesting that P450Hy01 is prominent candidate gene for P450-enzyme encoding 5EAl, 3H in pepper cell.

• BMB Reports
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• v.33 no.6
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• pp.519-524
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• 2000

NF1 proteins are a family of DNA binding proteins which consist of two separate domains, N-terminal DNA binding domain and C-terminal transcription activation domain. The N-terminal 220 amino acids are highly conserved and are also known to mediate dimerization of NF1 proteins. The C-terminal regions of different type of NF1 proteins are heterogeneous and responsible for transcriptional activation. In this study, we tested the interaction between different domains of rat NF1-A protein by yeast two hybrid analysis and observed the interaction between C-terminal regions of NF1-A which do not contain the N-terminal dimerization domain. Our results showed that the C-terminal region of rat NF1-A between residues 231 and 509 strongly interacted not only with itself, but also with human NF1/CTF1 which is a different type of NF1. When the C-terminal region was divided into two fragments, one from residue 231 to 447 and the other from 448 to 509, the two fragments were able to interact with the C-terminal region of NF1-A significantly. This indicates that both fragments contain independent interaction domains. Analysis of the interactions with alanine substituted fragments showed that substitutions of the heptasequence, SPTSPTY of NF1-A, affected interaction between NF1 proteins. Our results strongly suggest that C-terminal regions may also be important for the formation of homo- and heterodimers in addition to the N-terminal dimerization domain. Also, the heptasequence motif may play some roles in dimer formation.

• Korean Journal of Plant Resources
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• v.31 no.5
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• pp.531-537
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• 2018

This study was carried to survey distribution of the nucleotide polymorphisms in heme-binding (HB) domain, which is highly conserved region between 1,210 and 1,240 bp of cytochrome P450, in domestic garlic cultivars. 120 garlic cultivars collected from Korea were classified into seven HB domain variation based on the nucleotide sequence of the domain. Northern type garlic cultivars, collected from Kyungpook, Chungnam, Chungpook and Kangwon province, showed 51.3% of KP2 type nucleotide sequence, 5'-TTT/GGC/GGT/GGA/CGG/AGA/ATA/TGT/CCT/GGA-3' with coding amino acid FGGGRRICPG, 13.7% of KP1, 11.3% of CP, 8.8% of CM and 5% of KW2 types. Southern type cultivars, collected from Kyungnam province, showed 52.5% of KM type nucleotide sequence, 5'-TTT/GGC/GCA/GGA/CGG/AGA/ATT/TGT/CCT/GGA-3' with coding amino acid FGAGRRICPG, 22.5% of KP2, 5.0% of KW2 and 2,5% of CP type nucleotide sequence. These results showed that Korean garlics were cultivated in highly mixed condition even in the same region.

• Journal of Plant Biology
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• v.39 no.2
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• pp.85-92
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• 1996

Small GTP-binding proteins are divided into three major group: Ras, Rho and Ypt/Rab. They have the conserved regions designed G1 to G5 that are critical in GDP/GTP exchange, GTP-induced conformational change and GTP hydrolysis. We isolated and characterized genomic DNA or cDNAfragments encoding G1 to G3 domains of small GTP-binding protein Rab and Rho from several plant species using two different PCR-based cloning strategies. Seven rab DNA fragments were isolated from 4 different plants, mung-bean, tobacco, rice and pepper using two degenerate primers corresponding to the GTP-binding domain G1 and G3 in small GTP-binding proteins. The amino acid sequences among these rab DNA fragments and other known small GTP-binding proteins shows that they belong to the Ypt/Rab family. Six rho DNA fragments were isolated from 5 different plants, mung-bean, rice, Arabidopsis, Allium and Gonyaulax using the nested PCR method that involves four degenerate primers corresponding to the GTP-binding domain G1, G3 and G4. The rho DNA fragments cloned show more than 90% homology to each other. Sequence comparison between plant and other known Rho family genes suggests that they are closely related (67 to 82% amino acid identity). Sequence analysis and southern blot analysis of rab and rho in mung-bean suggest than thses genes are encoded by multigene family in mung-bean.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2003.10a
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• pp.5-5
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• 2003

The purple acid phosphatases comprise a family of binuclear metal-containing enzymes. The metal centre contains one ferric ion and one divalent metal ion. Spectroscopic studies of the monomeric, ${\sim}$36 kDa mammalian purple acid phosphatases reveal the presence of an Fe(III)Fe(II) centre in which the metals are weakly antiferromagnetically coupled, whereas the dimeric, ${\sim}$110 000 kDa plant enzymes contain either Fe(III)Zn(II) or Fe(III)Mn(II). The three dimensional structures of the red kidney bean and pig enzymes show very similar arrangements of the metal ligands but some significant differences beyond the immediate vicinity of the metals. In addition to the catalytic domain, the plant enzyme contains a second domain of unknown function. A search of sequence databases was undertaken using a sequence pattern which includes the conserved metal-binding residues in the plant and animal enzymes. The search revealed the presence in plants of a 'mammalian-type' low molecular weight purple acid phosphatase, a high molecular weight form in some fungi, and a homologue in some bacteria. The catalytic mechanism of the enzyme has been investigated with a view to understanding the marked difference in specificity between the Fe-Mn sweet potato enzyme, which exhibits highly efficient catalysis towards both activated and unactivated phosphate esters, and other PAPs, which hydrolyse only activated esters. Comparison of the active site structures of the enzymes reveal some interesting differences between them which may account for the difference. The implications fur understanding the physiological functions of the enzymes will be discussed.

• Journal of Applied Biological Chemistry
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• v.48 no.3
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• pp.113-119
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• 2005

O-methylation mediated by O-methyltransferases (OMTs) is a common modification in natural product biosynthesis and contributes to diversity of secondary metabolites. OMTs use phenylpropanoids, flavonoids, other phenolics and alkaloids as substrates, and share common domains for S-adenosyl-L-methionine (AdoMet) and substrate binding. We searched Arabiposis genome and found 17 OMTs genes (AtOMTs). AdoMet- and substrate-binding sites were predicted. AdoMet binding domain of AtOMTs is highly conserved, while substrate-binding domain is diverse, indicating use of different substrates. In addition, expressions of six AtOMT genes in response to UV and in different tissues were investigated using real-time quantitative reverse transcriptase-polymerase chain reaction. All the AtOMTs investigated were expressed under normal growth condition and most, except AtOMT10, were induced after UV illumination. AtOMT1 and AtOMT8 were expressed in all the tissues, whereas AtOMT10 showed flower-specific expression. Analysis of these AtOMT gene expressions could provide some clues on AtOMT involvement in the cellular processes.

• Journal of Microbiology and Biotechnology
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• v.25 no.10
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• pp.1648-1652
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• 2015

Azaphilone polyketides are synthesized by iterative non-reducing fungal polyketide synthases (NR-fPKSs) with a C-terminus reductive domain (-R). Several azaphilone biosynthetic gene clusters contain a putative serine hydrolase gene; the Monascus azaphilone pigment (MAzP) gene cluster harbors mppD. The MAzP productivity was significantly reduced by a knockout of mppD, and the MAzP NR-fPKS-R gene (MpPKS5) generated its product in yeast only when co-expressed with mppD. Site-directed mutations of mppD for conserved Ser/Asp/His residues abolished the product formation from the MpPKS5/mppD co-expression. MppD and its homologs are thus proposed as a new protein factor involved in the product formation of NR-fPKS-R.