Crystal structure of a plant ribonuclease, RNase LE

Ribonuclease LE (RNase LE) from cultured tomato (Lycopersicon esculentum) c ells is a member of the RNase T-2 family showing broad base specificity. Th e crystal structure of RNase LE has been determined at 1.65 Angstrom resolu tion. The structure consists of seven alpha-helices and seven beta-strands, belonging to an alpha + beta type structure. Comparison of the structure o f RNase LE with that of RNase Rh, a microbial RNase belonging to the RNase T-2 family, reveals that while the overall folding topologies are similar t o each other, major insertions and deletions are found at the N-terminal re gions. The structural comparison, an amino acid sequence alignment of the R Nase T-2 enzymes, and comparison of the disulfide-bonding pattern of these enzymes show that the structure of RNase LE shown here is the basic framewo rk of the animal/plant subfamily of RNase T-2 enzymes (including a self-inc ompatibility protein called S-RNase), and the structure of RNase Rh is that of the fungal subfamily of RNase T-2 enzymes (including RNase T-2). Subseq uently, we superposed the active-site of the RNase LE with that of RNase Rh and found that (1) His39, Trp42, His92, Glu93, Lys96, and His97 of RNase L E coincided exactly with His46, Trp49, His104, Glu105, Lys108, and His109, respectively, of RNase Rh, and (2) two conserved water molecules were found at the putative P-1 sites of both enzymes. These facts suggest that plant RNase LE has a very similar hydrolysis mechanism to that of fungal RNase Rh , and almost all the RNase T-2 enzymes widely distributed in various specie s share a common catalytic mechanism. A cluster of hydrophobic residues was found on the active-site face of the RNase LE molecule and two large hydro phobic pockets exist. These hydrophobic pockets appear to be base binding s ites mainly by hydrophobic interactions and are responsible for the base no n-specificity of RNase LE. (C) 2000 Academic Press.