• 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.

• Journal of Life Science
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• v.15 no.3 s.70
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• pp.415-422
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• 2005

library of the mutants was prepared by transposon mutagenesis of the Mycobacterium bovis BCG. We screened this library for the resistance to an anti-tuberculosis antibiotic, PA-824. Most of the mutants resistant to the PA-824 were not able to synthesize the coenzyme $F_{420}$ which is normally produced by the wild type M. bovis BCG strains. HPLC analysis of the cellular extract showed that one of those mutants which lost the ability to synthesize $F_{420}$ still produced F0. The insertion site of the transposon in this mutant was determined by an inverse PCR and the transposon was found to be inserted in the Rv2435c open reading frame (ORF). Rv2435c ORF is predicted to encode an 80.3 kDa protein. Rv2435c protein appears to be bound to the cytoplasmic membrane, its N-terminal present in the periplasm and C-terminal in the cytoplasm. The C-terminal portion of this protein is highly homologous with the adenylyl cyclases of both prokaryotes and eukaryotes. There are 15 ORFs which have homology with the class III AC proteins in the genome of the M. tuberculosis and M. bovis. Two of those, Rv1625c and Rv2435c, are highly homologous with the mammalian ACs. We cloned the cytoplasmic domain of the Rv2435c ORF and expressed it with six histidine residues attached on its C-terminal in Escherichia coli to find out if this protein is a genuine AC. Production of that protein in E. coli was proved by purifying the histidine-tagged protein by using the Ni-NTA resin. This protein, however, failed to complement the cya mutation in E. coli, indicating that this protein lacks the AC activity. All of the further attempts to convert this protein to a functional AC by a mutagenesis with UV or hydroxylamine, or construction of several different fusion proteins with Rv1625c failed. It is, therefore, possible that Rv2435c protein might affect the conversion of F0 to $F_{420}$ not by synthesizing cAMP but by some other way.

• Korean journal of applied entomology
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• v.43 no.2
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• pp.155-162
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• 2004

Serotonin receptor binds to serotonin (5-HT) and activates effector proteins such as adenylyl cyclase, phospholipase C, cyclic GMP phosphodiesterase or ion channel through G protein on the cell membrane, resulting in various physiological responses like diuresis, memory and development. To examine the comparative expression of the 5-HT$\_$7/ receptor of Aedes aegypti, the Aedes 5-HT$\_$7/ receptor gene was transfected into Drosophila Schneider2 (S2) cells and mammalian Chinese hamster ovary (CHO)-Kl cells. The expression of the Aedes 5-HT$\_$7/ receptor gene in selected cell lines, Tr-CHO and Tr-S2, was confirmed with reverse transcription-PCR, Western blot and immunocytochemistry. Compared with the induced intracellular cAMP level of Tr-S2 cell line to 5-HT, the induced cAMP in the Tr-CHO cell line was over 9 times higher and was dose-dependent. These results suggest that the functionality of Aedes 5-HT$\_$7/ receptor is much more effective in mammalian CHO-K 1 cells and that the Tr-CHO cell line expressing Aedes 5-HT$\_$7/ receptor can be used for synthetic agonist or antagonist candidate screening.

• Journal of Life Science
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• v.23 no.5
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• pp.609-615
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• 2013

Glyceollin I has gained attention as a useful therapy for various dermatological diseases. However, the binding property of glyceollin I to the mammalian adenylyl cyclase (hereafter mAC), a critical target enzyme for the down-regulation of skin melanogenesis, has not been fully explored. To clarify the action mechanism between glyceollin I and mAC, we first investigated the molecular docking property of glyceollin I to mAC and compared with that of SQ22,536, a well-known mAC inhibitor, to mAC. Glyceollin I showed superiority by forming three hydrogen bonds with Asp 1018, Trp 1020, and Asn 1025, which exist in the catalytic site of mAC. However, SQ22,536 formed only two hydrogen bonds with Asp 1018 and Asn 1025. Secondly, we confirmed that glyceollin I effectively inhibits the formation of forskolin-induced cAMP and the phosphorylation of PKA from a cell-based assay. Long term treatment with glyceollin I had little effect on the cell viability. The findings of the present study also suggest that glyceollin I may be extended to be used as an effective inhibitor of hyperpigmentation.