The Base Specificities of Tomato Ribonuclease (RNase LE) and Its Asp44 Mutant Enzyme Expressed from Yeast Cells

Abstract

RNase LE from cultured tomato cells is a member of the RNase T₂ family. It is, however, distinguishable from RNase Rh from Rhizopus niveus, a typical RNase of the RNase T₂ family, by its CD spectrum in the 200-250 nm region. In order to reinvestigate the base specificity of RNase LE and to study the role of Asn44 in RNase LE, which is considered to correspond to the base recognition site Asp51 of RNase Rh, RNase LE, and its Asp mutant at the 44th position were expressed from yeast cells with the same expression system as RNase Rh [K. Ohgi, et al., L Biochem., 109, 776-785 (1991)]. RNase LE with four extra amino acid residues at the 2nd amino acid residue of mature RNase LE and its Asp44 mutant were secreted from yeast cells to give a yield of 10 mg/liter and 0.5 mg/liter culture broth, respectively. The expressed RNase LE (RNase RNAP LE) had the same characteristics as native RNase LE in the CD spectrum and specitic activity. This is the first example of the expression of plant RNase from microbes and in sufficient amount to perform further enzymological research. The base specificity of RNase LE was guanylic acid preferential and that of N44D was changed to a more adenylic acid preference as compared to that of RNase LE. These experiments showed that Asn44 of RNase LE is crucial for base recognition as the case of Asp51 in RNase Rh, and also suggested that the base recognition methanism of RNase LE is very similar to that of RNase Rh.