Inhibitory effects on mushroom tyrosinase by flavones from the stem barks of Morus lhou (S.) Koidz.

Five flavones displaying tyrosinase inhibitory activity were isolated from the stem barks of Morus lhou (S.) Koidz., a cultivated edible plant. The isolated compounds were identified as mormin (1), cyclomorusin (2), morusin (3), kuwanon C (4), and norartocarpetin (5). Mormin (1) was characterized as a new flavone possesing a 3-hydroxymethyl-2-butenyl at C-3. The inhibitory potencies of these flavonoids toward monophenolase activity of mushroom tyrosinase were investigated. The IC50 values of compounds 1-5 for monophenolase activity were determined to be 0.088, 0.092, 0.250, 0.135 mM, and 1.2 microM, respectively. Mormin (1), cyclomorusin (2), kuwanon C (4) and norartocarpetin (5) exhibited competitive inhibition characteristics. Interestingly norartocarpetin (5) showed a time-dependent inhibition against oxidation of L-tyrosine: it also operated under the enzyme isomerization model (k5 = 0.8424 min(-1), k6 = 0.0576 min(-1), K(app)(i) = 1.354 microM).

A new class II rice chitinase, Rcht2, whose induction by fungal elicitor is abolished by protein phosphatase 1 and 2A inhibitor.

Among the four classes of chitinase, a class II chitinase had not yet been reported for rice. We have isolated and characterized a class II acidic chitinase, Rcht2, from rice (Oryza sativa L. cv. Cheongcheongbyeo). The protein consists of a single polypeptide chain of 261 amino acid residues and includes a putative signal sequence of 29 amino acids at its N-terminus. It has a calculated molecular mass of 27,642 Da and an isoelectric point of 5.56. The Rcht2 chitinase lacks the cysteine-rich and hinge domains in the N-terminal region of the protein, which is the criterion for its classification as a class II chitinase. Comparison of the genomic and the cDNA sequence revealed that the coding region of Rcht2 consist of three exons of 301, 112, and 370 bp separated by two introns of 89 and 984 bp. In suspension-cultured rice cells, the transcript level of Rcht2 was dramatically increased by treatment with both glycol chitin and fungal elicitor. The application of protein phosphatase 1 and 2A inhibitors, calyculin A and okadaic acid, effectively abolished the induction of Rcht2 in response to fungal elicitor. In contrast, the activation of Rcht2 transcript was not inhibited by both cycloheximide and protein kinase inhibitors. These results demonstrate that protein dephosphorylation events play a crucial role in the elicitor-mediated induction of Rcht2 in rice cells, while de novo protein synthesis is not required for induction.

Selection of a RNA aptamer that binds to human activated protein C and inhibits its protease function.

A high-affinity RNA aptamer to human activated protein C (APC) was selected from a pool of random sequences using in vitro selection. Activated protein C, a trypsin-like serine protease plays an important role along with thrombin as a regulator in blood clotting cascade. After seven rounds of selection and amplification, a single predominant nucleic acid sequence APC-167, a 167-base oligonucleotide with a random sequence core of 120 bases, was obtained. The selected aptamer did not bind to thrombin or factor Xa and thus demonstrated specificity to APC. Furthermore, this aptamer was a non-competitive inhibitor to the cleavage reaction of a fluorogenic substrate catalyzed by APC. The inhibition constant (Ki) of APC-167 was 83 nM. The 99-base oligonucleotide (APC-99) derived from APC-167 by deleting both primer binding sites, was also found to inhibit APC strongly (Ki = 137 nM). Two stem-loop structures and at least one G x U wobble base pair in the stem were elucidated as important structural motifs for binding.

Cloning of the 52-kDa chitinase gene from Serratia marcescens KCTC2172 and its proteolytic cleavage into an active 35-kDa enzyme.

A chitinase gene (pCHI52) encoding the 52-kDa chitinase was isolated from a Serratia marcescens KCTC2172 cosmid library. This chitinase gene consists of 2526 bp with an open reading frame that encodes 485 amino acids. Escherichia coli harboring the pCHI52 gene secreted not only a 52-kDa but also a 35-kDa chitinase into the culture supernatant. We purified both 52-kDa and 35-kDa chitinases using a chitin affinity column and Sephacryl-S-300 gel filtration chromatography. We determined that the 17 N-terminal amino acid sequences of the 52-kDa and the 35-kDa chitinase are identical. Furthermore, a protease obtained from S. marcescens KCTC2172 cleaved the 52-kDa chitinase into the 35-kDa protein with chitinase activity. These results suggest that the 35-kDa chitinase derives from the 52-kDa chitinase by post-translational proteolytic modification. The optimal reaction temperature of 45 degrees C and the optimal pH of 5.5 were identical for both enzymes. The specific activities of the 52-kDa and 35-kDa chitinases on natural swollen chitin were 67 mumol min-1 mg-1 and 60 mumol min-1 mg-1, respectively.

Isolation and characterization of the 54-kDa and 22-kDa chitinase genes of Serratia marcescens KCTC2172.

A DNA fragment (pCHI5422) containing two genes encoding a 54-kD and a 22-kDa chitinase was isolated from a cosmid DNA library of Serratia marcescens KCTC2172. The complete nucleotide sequence of pCHI5422 consisting of 4581 bp was determined. The nucleotide sequence of the 22-kDa chitinase consists of 681 bp of open reading frame encoding 227 amino acids and is located 1422 bp downstream of the translation termination codon of the 54-kDa chitinase sequence. The 54-kDa chitinase gene consisted of 1497 bp in a single open reading frame encoding 499 amino acids. The genes encoding the 54-kDa and 22-kDa chitinase were separately subcloned in Escherichia coli and the individual chitinases were expressed and purified from the culture broth using chitin affinity chromatography. When chitohexaose was used as substrate, the major product of the enzymatic reaction of both the 54-kDa and 22-kDa chitinases was a (GlcNAc)2 dimer with a minor amount of monomer. The specific activity of the 54-kDa and 22-kDa chitinases were 300 microM (min)-1 mg-1 and 17 microM (min)-1 mg-1 on the natural swollen chitin, respectively.

Overproduction of indole acetic acid in Azospirillum lipoferum using the Escherichia coli trp operon.

A recombinant plasmid carrying the trp operon from Escherichia coli, which synthesizes tryptophan from chorismate, was constructed by using a broad host range plasmid vector pRK290; a mutant trp plasmid for tryptophan overproduction was then selected. The physiological, biochemical, and genetic properties of the Azospirillum lipoferum KY6, a potential nitrogen fixer of rice, harbouring the recombinant trp plasmid pMJC1 and its mutant pMJC101, were compared with those of the wild-type bacteria. Anthranilate synthetase is known to be the trpE gene product which plays a key role in the regulatory step in the feedback control of tryptophan biosynthesis. The enzyme activity of the Azospirillum lipoferum KY6 carrying pMJC1 or pMJC101 was respectively 7- and 30-fold higher than that of the wild type in the presence of 10(-4)M tryptophan. As expected, the amount of tryptophan biosynthesis in A. lipoferum KY6 (pMJC101) was increased approximately 100-fold as compared with the wild type, which led to overproduction of indole acetic acid even without addition of exogenous tryptophan. Moreover, the recombinant trp plasmid was fairly stable in A. lipoferum KY6 host, showing only 25% loss of the plasmid itself or the trp insert after 40 generations.