Ribonuclease NW (
RNase NW), the
wound-inducible
RNase in Nicotiana glutinosa leaves, preferentially cleaves
guanylic acid. We expressed the
cDNA encoding
RNase NW in the methylotrophic yeast Pichia pastoris using the expression vector pPIC9K, and the resulting recombinant
RNase NW (ryRNaseNW) secreted into medium was purified to apparent homogeneity using column chromatography. The crystal structure of ryRNase NW bound to
5'-GMP was determined at 1.5 A resolution by molecular replacement with tomato
RNase LE as a search model. The
RNase NW structurally belongs to the (alpha + beta) class of
proteins, having eight helices (five alpha-helices and three 3(10) helices) and six beta-strands, and its structure is highly similar to those of other plant RNases, including a
uridylic acid preferential
RNase MC1 from bitter gourd seeds. The
guanine ring of
5'-GMP lies in a hydrophobic pocket of the molecular surface composed of Tyr17, Tyr71, Ala80, Leu79, and Phe89: the
guanine base is sandwiched between aromatic side chains of Tyr17 and Phe89. In addition, the
guanine base is firmly stabilized by a network of hydrogen bonds of the side chains of Gln12 and Thr78, as well as of the main chain of Leu79. Therefore, Gln12, Tyr17, Thr78, Leu79, and Phe89 are responsible for recognition of the
guanine base by
RNase NW, findings which provide insight into the manner in which
RNase NW preferentially cleaves
guanylic acid.