• Journal of Microbiology and Biotechnology
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• 제26권12호
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• pp.2087-2097
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• 2016

Most cold-adapted enzymes possess higher $K_m$ and $k_{cat}$ values than those of their mesophilic counterparts to maximize the reaction rate. This characteristic is often ascribed to a high structural flexibility and improved dynamics in the active site. However, this may be less convincing to cold-adapted metabolic enzymes, which work at substrate concentrations near $K_m$. In this respect, cold adaptation of a shikimate kinase (SK) in the shikimate pathway from psychrophilic Colwellia psychrerythraea (CpSK) was characterized by comparing it with a mesophilic Escherichia coli homolog (EcSK). The optimum temperatures for CpSK and EcSK activity were approximately $30^{\circ}C$ and $40^{\circ}C$, respectively. The melting points were $33^{\circ}C$ and $45^{\circ}C$ for CpSK and EcSK, respectively. The ${\Delta}G_{H_2O}$ (denaturation in the absence of denaturing agent) values were 3.94 and 5.74 kcal/mol for CpSK and EcSK, respectively. These results indicated that CpSK was a cold-adapted enzyme. However, contrary to typical kinetic data, CpSK had a lower $K_m$ for its substrate shikimate than most mesophilic SKs, and the $k_{cat}$ was not increased. This observation suggested that CpSK may have evolved to exhibit increased substrate affinity at low intracellular concentrations of shikimate in the cold environment. Sequence analysis and homology modeling also showed that some important salt bridges were lost in CpSK, and higher Arg residues around critical Arg 140 seemed to increase flexibility for catalysis. Taken together, these data demonstrate that CpSK exhibits characteristics of cold adaptation with unusual kinetic parameters, which may provide important insights into the cold adaptation of metabolic enzymes.

• Bulletin of the Korean Chemical Society
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• 제29권7호
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• pp.1311-1314
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• 2008

Bacillus halodurans O-methyltransferase (BhOMT) is an S-adenosylmethionine dependent methyltransferase. In our previous study, three dimensional structure of the BhOMT has been determined by comparative homology modeling and automated docking study showed that two hydroxyl groups at 3'- and 4'-position in Bring and structural rigidity of C-ring resulting from the double bond characters between C2 and C3 of flavonoid, were key factors for interaction with BhOMT. In the present study, BhOMT was cloned and expressed. Binding assay was performed on purified BhOMT using fluorescence experiments and binding affinity of luteolin, quercetin, fisetin, and myricetin were measured in the range of $10^7$. Fluorescence quenching experiments indicated that divalent cation plays a critical role on the metal-mediated electrostatic interactions between flavonoid and substrate binding site of BhOMT. Fluorescence study confirmed successfully the data obtained from the docking study and these results imply that hydroxyl group at 7-position of luteolin, quercetin, fisetin, and myricetin forms a stable hydrogen bonding with K211 and carboxyl oxygen of C-ring forms a stable hydrogen bonding with R170. Hydroxyl group at 3'-and 4'-position in the B-ring also has strong $Ca^{2+}$ mediated electrostatic interactions with BhOMT.

• Journal of Microbiology and Biotechnology
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• 제30권11호
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• pp.1750-1759
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• 2020

The characterization of cytochrome P450 CYP125A13 from Streptomyces peucetius was conducted using cholesterol as the sole substrate. The in vitro enzymatic assay utilizing putidaredoxin and putidaredoxin reductase from Pseudomonas putida revealed that CYP125A13 bound cholesterol and hydroxylated it. The calculated K_D value, catalytic conversion rates, and Km value were 56.92 ± 11.28 μM, 1.95 nmol min^-1 nmol^-1, and 11.3 ± 2.8 μM, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis showed that carbon 27 of the cholesterol side-chain was hydroxylated, characterizing CYP125A13 as steroid C27-hydroxylase. The homology modeling and docking results also revealed the binding of cholesterol to the active site, facilitated by the hydrophobic amino acids and position of the C27-methyl group near heme. This orientation was favorable for the hydroxylation of the C27-methyl group, supporting the in vitro analysis. This was the first reported case of the hydroxylation of cholesterol at the C-27 position by Streptomyces P450. This study also established the catalytic function of CYP125A13 and provides a solid basis for further studies related to the catabolic potential of Streptomyces species.

• Genomics & Informatics
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• 제3권4호
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• pp.142-148
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• 2005

The immune response-related genes have been suggested to be the most favorable genes for positive selection during evolution. Comparing the entire DNA sequence of chimpanzee chromosome 22 (PTR22) with human chromosome 21 (HSA21), we have identified 15 orthologs having indel in their coding sequences. Among them, interferon-${\alpha}$ receptor-1 gene (IFNAR1), an immuneresponse-related gene, is subjected to comparative genomic analysis. Chimpanzee IFNAR1 showed the same genomic structure as human IFNAR1 (11 exons and 10 introns) except the 3 bp insertion in exon 4. The sequence alignment of IFNAR1 coding sequence indicated that 'ISPP' amino acid sequence motif is highly conserved in chimpanzee and other animals including mouse and chicken. However, the human IFNAR1 shows that one proline residue is missing in the sequence motif. The homology modeling of the IFNAR1 structures suggests that the proline deletion in human IFNAR1 leads to the formation of the following ${\alpha}$-helix, whereas two sequential prolines in chimpanzee IFNAR1 inhibit it. As a result, human IFNAR1 may adopt a characteristic structure distinct from chimpanzee IFNAR1. This human specific trait could contribute to specific immune response in the most optimized manner for humans. Further molecular biological studies on the IFNAR1 will help us to gain insights into the molecular implication of species-specific host-pathogen interaction in primate evolution.

• Journal of Ginseng Research
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• 제39권4호
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• pp.406-413
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• 2015

Background: Pathogenesis-related 10 (PR-10) proteins are small, cytosolic proteins with a similar three-dimensional structure. Crystal structures for several PR-10 homologs have similar overall folding patterns, with an unusually large internal cavity that is a binding site for biologically important molecules. Although structural information on PR-10 proteins is substantial, understanding of their biological function remains limited. Here, we showed that one of the PgPR-10 homologs, PgPR-10.3, shares binding properties with flavonoids, kinetin, emodin, deoxycholic acid, and ginsenoside Re (1 of the steroid glycosides). Methods: Gene expression patterns of PgPR-10.3 were analyzed by quantitative real-time PCR. The three-dimensional structure of PgPR-10 proteins was visualized by homology modeling, and docking to retrieve biologically active molecules was performed using AutoDock4 program. Results: Transcript levels of PgPR-10.3 expressed in leaves, stems, and roots of 3-wk-old ginseng plantlets were on average 86-fold lower than those of PgPR-10.2. In mature 2-yr-old ginseng plants, the mRNA of PgPR-10.3 is restricted to leaves. Ginsenoside Re production is especially prominent in leaves of Panax ginseng Meyer, and the binding property of PgPR-10.3 with ginsenoside Re suggests that this protein has an important role in the control of secondary metabolism. Conclusion: Although ginseng PR-10.3 gene is expressed in all organs of 3-wk-old plantlets, its expression is restricted to leaves in mature 2-yr-old ginseng plants. The putative binding property of PgPR-10.3 with Re is intriguing. Further verification of binding affinity with other biologically important molecules in the large hydrophobic cavity of PgPR-10.3 may provide an insight into the biological features of PR-10 proteins.

• Journal of Microbiology and Biotechnology
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• 제31권3호
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• pp.483-491
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• 2021

Two putative genes, lip29 and est29, encoding lipolytic enzymes from the thermophilic bacterium Geobacillus thermocatenulatus KCTC 3921 were cloned and overexpressed in Escherichia coli. The recombinant Lip29 and Est29 were purified 67.3-fold to homogeneity with specific activity of 2.27 U/mg and recovery of 5.8% and 14.4-fold with specific activity of 0.92 U/mg and recovery of 1.3%, respectively. The molecular mass of each purified enzyme was estimated to be 29 kDa by SDS-PAGE. The alignment analysis of amino acid sequences revealed that both enzymes belonged to GDSL lipase/esterase family including conserved blocks with SGNH catalytic residues which was mainly identified in plants before. While Est29 showed high specificity toward short-chain fatty acids (C4-C8), Lip29 showed strong lipolytic activity to long-chain fatty acids (C12-C16). The optimal activity of Lip29 toward p-nitrophenyl palmitate as a substrate was observed at 50℃ and pH 9.5, respectively, and its activity was maintained more than 24 h at optimal temperatures, indicating that Lip29 was thermostable. Lip29 exhibited high tolerance against detergents and metal ions. The homology modeling and substrate docking revealed that the long-chain substrates showed the greatest binding affinity toward enzyme. Based on the biochemical and insilico analyses, we present for the first time a GDSL-type lipase in the thermophilic bacteria group.

• Genomics & Informatics
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• 제18권3호
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• pp.28.1-28.9
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• 2020

Streptomyces coelicolor is a gram-positive soil bacterium which is well known for the production of several antibiotics used in various biotechnological applications. But numerous proteins from its genome are considered hypothetical proteins. Therefore, the present study aimed to reveal the functions of a hypothetical protein from the genome of S. coelicolor. Several bioinformatics tools were employed to predict the structure and function of this protein. Sequence similarity was searched through the available bioinformatics databases to find out the homologous protein. The secondary and tertiary structure were predicted and further validated with quality assessment tools. Furthermore, the active site and the interacting proteins were also explored with the utilization of CASTp and STRING server. The hypothetical protein showed the important biological activity having with two functional domain including POD-like_MBL-fold and rhodanese homology domain. The functional annotation exposed that the selected hypothetical protein could show the hydrolase activity. Furthermore, protein-protein interactions of selected hypothetical protein revealed several functional partners those have the significant role for the bacterial survival. At last, the current study depicts that the annotated hypothetical protein is linked with hydrolase activity which might be of great interest to the further research in bacterial genetics.

• Molecules and Cells
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• 제46권8호
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• pp.513-525
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• 2023

Orange carotenoid protein (OCP) of photosynthetic cyanobacteria binds to ketocarotenoids noncovalently and absorbs excess light to protect the host organism from light-induced oxidative damage. Herein, we found that mutating valine 40 in the α3 helix of Gloeocapsa sp. PCC 7513 (GlOCP1) resulted in blue- or red-shifts of 6-20 nm in the absorption maxima of the lit forms. We analyzed the origins of absorption maxima shifts by integrating X-ray crystallography, homology modeling, molecular dynamics simulations, and hybrid quantum mechanics/molecular mechanics calculations. Our analysis suggested that the single residue mutations alter the polar environment surrounding the bound canthaxanthin, thereby modulating the degree of charge transfer in the photoexcited state of the chromophore. Our integrated investigations reveal the mechanism of color adaptation specific to OCPs and suggest a design principle for color-specific photoswitches.

• Applied Microscopy
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• 제51권
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• pp.17.1-17.10
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• 2021

Our previous study on the binding activity between Cel5H and clay minerals showed highest binding efficiency among other cellulase enzymes cloned. Here, based on previous studies, we hypothesized that the positive amino acids on the surface of Cel5H protein may play an important role in binding to clay surfaces. To examine this, protein sequences of Bacillus licheniformis Cel5H (BlCel5H) and Paenibacillus polymyxa Cel5A (PpCel5A) were analyzed and then selected amino acids were mutated. These mutated proteins were investigated for binding activity and force measurement via atomic force microscopy (AFM). A total of seven amino acids which are only present in BlCel5H but not in PpCel5A were selected for mutational studies and the positive residues which are present in both were omitted. Of the seven selected surface lysine residues, only three mutants K196A(M2), K54A(M3) and K157T(M4) showed 12%, 7% and 8% less clay mineral binding ability, respectively compared with wild-type. The probable reason why other mutants did not show altered binding efficiency might be due to relative location of amino acids on the protein surface. Meanwhile, measurement of adhesion forces on mica sheets showed a well-defined maximum at 69±19 pN for wild-type, 58±19 pN for M2, 53±19 pN for M3, and 49±19 pN for M4 proteins. Hence, our results demonstrated that relative location of surface amino acids of Cel5H protein especially positive charged amino acids are important in the process of clay mineral-protein binding interaction through electrostatic exchange of charges.

• 생물정신의학
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• 제30권1호
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• pp.17-23
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• 2023

Objectives This study aims to develop valid experimental models for depression through chronic reserpine exposure to zebrafish (Danio rerio). Methods The effect of chronic reserpine on zebrafish behavior in the novel tank was examined. Changes of gene expression on telencephalon were also investigated. Results Chronic reserpine (40 mg/L, 7 days) induced overt behavioral effects, but markedly reduced activity, resembling motor retardation in depression. In telencephalon of zebrafish, gene expression associated with monoamine oxidase and norepinephrine transporter was decreased. Expression of serotonin transporter gene was increased. Conclusions Our results show that the pharmacological model of depression in zebrafish can induce not only behavioral changes, but also monoamine changes in the homology of human mood regulation centers.