- Identification of Essential Amino acid Residues in Valine Dehydrogenase from Streptomyces albus
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Chang-Gu Hyun , Sang-Suk Kim , Joo-Won Suh
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J. Microbiol. 2006;44(1):50-53.
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DOI: https://doi.org/2337 [pii]
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 Abstract
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Cys-29 and Cys-251 of Streptomyces albus valine dehydrogenase (ValDH) were highly conserved
in the corresponding region of NAD(P)+-dependent amino acid dehydroganase sequences. To ascertain
the functional role of these cysteine residues in S. albus ValDH, site-directed mutagenesis
was performed to change each of the two residues to serine. Kinetic analyses of the enzymes mutated
at Cys-29 and Cys-251 revealed that these residues are involved in catalysis. We also constructed
mutant ValDH by substituting valine for leucine at 305 by site-directed mutagenesis. This
residue was chosen, because it has been proposed to be important for substrate discrimination by
phenylalanine dehydrogenase (PheDH) and leucine dehydrogenase (LeuDH). Kinetic anaysis of the
V305L mutant enzyme revealed that it is involved in the substrate binding site. However it displayed
less activity than the wild type enzyme toward all aliphatic and aromatic amino acids
tested.
- Molecular Detection of [alpha]-Glucosidase Inhibitor-producing Actinomycetes
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Chang-Gu Hyun , Seung-Young Kim , Jin-Haeng Hur , Myung-Ji Seo , Joo-Won Suh , Soon-Ok Kim
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J. Microbiol. 2005;43(3):313-318.
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DOI: https://doi.org/2207 [pii]
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Abstract
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In this study, we demonstrate the use of a PCR-based method for the detection of the specific genes involved in natural-product biosynthesis. This method was applied, using specifically designed PCR primers, to the amplification of a gene segment encoding for sedo-heptulose 7-phosphate cyclase, which appears to be involved in the biosynthetic pathways of C_7N aminoacyclitol or its keto analogue-containing metabolites, in a variety of actinomycetes species. The sequences of DNA fragments (about 540 bp) obtained from three out of 39 actinomycete strains exhibited a high degree of homology with the sedo-heptulose 7-phosphate cyclase gene, which has been implicated in acarbose biosynthesis. The selective cultivation conditions of this experiment induced the expression of these loci, indicating that the range of C_7N aminoacyclitol or its keto analogue-group natural products might be far greater than was previously imagined. Considering that a total of approximately 20 C_7N aminoacyclitol metabolites, or its keto analogue-containing metabolites, have been described to date, it appears likely that some of the unknown loci described herein might constitute new classes of C_7N aminoacyclitol, or of its keto analogue-containing metabolites. As these metabolites, some of which contain valienamine, are among the most potent antidiabetic agents thus far discovered, the molecular detection of specific metabolite-producing actinomycetes may prove a crucial step in current attempts to expand the scope and diversity of natural-product discovery.
- Molecular Cloning and Analysis of the Gene for P-450 Hydroxylase from Pseudonocardia autotrophica IFO 12743
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Jung-Mee Kim , Younmie Jin , Chang-Gu Hyun , Jong-Hee Kim , Hong-Sub Lee , Dae-Kyung Kang , Dae-Jung Kang , Tae-Yong Kim , Joo-Won
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J. Microbiol. 2002;40(3):211-218.
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Abstract
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A 4.8-kb DNA fragment encoding the P-450 type hydroxylase and ferredoxin genes was cloned from Pseudonocardia autotrophica IFO 12743 that can convert vitamin D_3 into its hydroxylated active forms. In order to isolate the P-450 gene cluster in this organism, we designed PCR primers on the basis of the regions of an oxygen binding site and a heme ligand pocket that are general characteristics of the P-450 hydroxylase. Sequencing analysis of the BamHI fragment revealed the presence of four complete and one incomplete ORFs, named PauA, PauB, PauC, and PauD, respectively. As a result of computer-based analyses, PauA and PauB have homology with enoyl-CoA hydratase from several organisms and the positive regulators belonging to the tetR family, respectively. PauC and PauD show similarity with SuaB/C proteins and ferredoxins, respectively, which are composed of P-450 monooxygenase systems for metabolizing two sulfonylurea herbicides in Streptomyces griseolus PauC shows the highest similarity with another CytP-450_Sca2 protein that is responsible for production of a specific HMG-CoA reductase inhibitor, pravastatin, in S. carbophilus. Cultures of Streptomyces lividans transformant, containing the P-450 gene cluster on the pWHM3 plasmid, was unable to convert vitamin D_3 to its hydroxylated forms.
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