Reactivating chaperones for coenzyme B-dependent diol and glycerol dehydratases and ethanolamine ammonia-lyase.

Adenosylcobalamin (AdoCbl) or coenzyme B-dependent enzymes tend to undergo mechanism-based inactivation during catalysis or inactivation in the absence of substrate. Such inactivation may be inevitable because they use a highly reactive radical for catalysis, and side reactions of radical intermediates result in the damage of the coenzyme. How do living organisms address such inactivation when enzymes are inactivated by undesirable side reactions? We discovered reactivating factors for radical B eliminases.

Coenzyme B-dependent eliminases: Diol and glycerol dehydratases and ethanolamine ammonia-lyase.

Adenosylcobalamin (AdoCbl) or coenzyme B-dependent enzymes catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. They use a super-reactive primary-carbon radical formed by the homolysis of the coenzyme's Co-C bond for catalysis and thus belong to the larger class of "radical enzymes." For understanding the general mechanisms of radical enzymes, it is of great importance to establish the general mechanism of AdoCbl-dependent catalysis using enzymes that catalyze the simplest reactions-such as diol dehydratase, glycerol dehydratase and ethanolamine ammonia-lyase.

Necessity of Flanking Repeats R1' and R8' of Human Pumilio1 Protein for RNA Binding.

Human Pumilio (hPUM) is a structurally well-analyzed RNA-binding protein that has been used recently for artificial RNA binding. Structural analysis revealed that amino acids at positions 12, 13, and 16 in the repeats from R1 to R8 each contact one specific RNA base in the eight-nucleotide RNA target. The functions of the N- and C-terminal flanking repeats R1' and R8', however, remain unclear.

Genome sequence analysis of new plum pox virus isolates from Japan.

Objective: To find mutations that may have recently occurred in Plum pox virus (PPV), we collected six PPV-infected plum/peach trees from the western part of Japan and one from the eastern part. After sequencing the full-length PPV genomic RNAs, we compared the amino acid sequences with representative isolates of each PPV strain.

Results: All new isolates were found to belong to the PPV-D strain: the six isolates collected from western Japan were identified as the West-Japan strain while the one collected from eastern Japan as the East-Japan strain.

Site-Specific Integration by Recruitment of a Complex of ΦC31 Integrase and Donor DNA to a Target Site by Using a Tandem, Artificial Zinc-Finger Protein.

To solve the problem of uncontrolled therapeutic gene integration, which is a critical drawback of retroviral vectors for gene therapy, the integration sites of exogenous genes should be precisely controlled not to perturb endogenous gene expression. To accomplish this, we explored the possibility of site-specific integration using two six-finger artificial zinc-finger proteins (AZPs) tandemly conjugated via a flexible peptide linker (designated "Tandem AZP"). A Tandem AZP in which two AZPs recognize specific 19 bp targets in a donor and acceptor DNA was expected to site-specifically recruit the donor DNA to the acceptor DNA.