ADP-ribosylation of guanosine by SCO5461 protein secreted from Streptomyces coelicolor.

The Streptomyces coelicolor A3(2) genome encodes a possible secretion protein, SCO5461, that shares a 30% homology with the activity domains of two toxic ADP-ribosyltransferases, pierisins and mosquitocidal toxin. We found ADP-ribosylating activity for the SCO5461 protein product through its co-incubation with guanosine and NAD(+), which resulted in the formation of N(2)-(ADP-ribos-1-yl)-guanosine ((ar2)Guo), with a K(m) value of 110 µM. SCO5461 was further found to ADP-ribosylate deoxyguanosine, GMP, dGMP, GTP, dGTP, and cyclic GMP with k(cat) values of 150-370 s(-1). Oligo(dG), oligo(G), and yeast tRNA were also ADP-ribosylated by this protein, although with much lower k(cat) values of 0.2 s(-1) or less. SCO5461 showed maximum ADP-ribosylation activity towards guanosine at 30 °C, and maintained 20% of these maximum activity levels even at 0 °C. This is the first report of the ADP-ribosylation of guanosine and guanine mononucleotides among the family members of various ADP-ribosylating enzymes. We additionally observed secretion of the putative gene product, SCO5461, in liquid cultures of S. coelicolor. We thus designated the SCO5461 protein product as S. coelicolor ADP-ribosylating protein, ScARP. Our current results could offer new insights into not only the ADP-ribosylation of small molecules but also signal transduction events via enzymatic nucleoside modification by toxin-related enzymes.

Molecular cloning of apoptosis-inducing Pierisin-like proteins, from two species of white butterfly, Pieris melete and Aporia crataegi.

Pierisin-1, present in cabbage butterfly, Pieris rapae, induces apoptosis against various kinds of cancer cell lines. Another cabbage butterfly, Pieris brassicae, also has an apoptosis-inducing protein, Pierisin-2. These proteins exhibit DNA ADP-ribosylating activity. Pierisin-like proteins are found to be distributed in subtribes Pierina, Aporiina and Appiadina. In this study, we performed the cDNA cloning of Pierisin-like proteins designated Pierisin-3 from gray-veined white, Pieris melete, and Pierisin-4 from black-veined white, Aporia crataegi. The nucleotide sequences of Pierisin-3 and -4 encode an 850 and an 858 amino acid protein, respectively. The partial peptide sequences of Pierisin-3 and -4 purified from pupae were identical to the deduced amino acid sequence of ORF. The deduced amino acid sequence revealed that Pierisin-3 is 93% similar to Pierisin-1 and Pierisin-4 is 64%. Pierisin-3 and -4 synthesized in vitro with the rabbit reticulocyte lysate exhibited apoptosis-inducing activity against human cervical carcinoma HeLa and human gastric carcinoma TMK-1 cells. Site-directed mutagenesis at a glutamic acid residue comprising the NAD-binding site resulted in a significant decrease in cytotoxicity of both proteins. Moreover, the proteins incubated with calf thymus DNA and beta-NAD resulted in the formation of N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine, as in the case of Pierisin-1 and -2. These findings could provide useful information for understanding the importance of apoptosis-inducing ability and molecular evolution of Pierisin-like proteins in family Pieridae.

Enzymatic properties of pierisin-1 and its N-terminal domain, a guanine-specific ADP-ribosyltransferase from the cabbage butterfly.

The cabbage butterfly, Pieris rapae, produces an ADP-ribosylating cytotoxic protein, pierisin-1. Unlike other ADP-ribosylating toxins, the acceptor site for ADP-ribosylation by pierisin-1 is the N-2 position of guanine bases in DNA. The present study was designed to characterize this novel guanine-specific ADP-ribosyltransferase, pierisin-1. The N-terminal polypeptide from Met-1 to Arg-233, but not the C-terminal Ser-234-Met-850 polypeptide, was found to exhibit guanine ADP-ribosyltransferase activity. Trypsin-treated pierisin-1, which is considered to be a "nicked" full-length form composed of associated N- and C-terminal fragments, also demonstrated such activity. Optimum conditions for the N-terminal polypeptide of pierisin-1 were pH 8-10, 37-40 degrees C, in the presence of 100-200 mM NaCl or KCl. Other metal ions such as Ca(2+) or Mg(2+) were not required. Kinetic studies demonstrated potent ADP-ribosyltransferase activity with a K(M) value for NAD of 0.17 mM and k(cat) of 55 per second. Under these optimum conditions, the specific activity of trypsin-treated pierisin-1 was about half (k(cat) = 25 per second). When the conditions were changed to pH 5-7 or 10-20 degrees C, some activity (6-55% or 5-20%, respectively, of that under optimal conditions) of the N-terminal polypeptide was still evident; however, almost all of the trypsin-treated enzyme activity disappeared. This implies the inhibition of the N-terminal enzyme domain by the associated C-terminal fragment. Long-term reactions indicated that a single molecule of pierisin-1 has the capacity to generate more than 10(6) ADP-ribosylated DNA adducts, which could cause the death of a mammalian cell.