The crystal structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus, has been determined at 2.5 Å resolution by the multiple isomorphous replacement method. The crystal structure was refined by simulated annealing with molecular dynamics. The current crystallographic R-factor is 0.200 in the 10—2.5 Å resolution range. The molecular structure which is completely different from the known structures of RNase A and RNase T1 consists of six α-helices and seven β-strands, belonging to the α+β type structure. Two histidine and one glutamic acid residues which were predicted as the most probably functional residues by chemical modification studies are found to be clustered. The steric nature of the active site taken together with the relevant site-directed mutagenesis experiments (Irie et al.) indicates that: (i) the two histidine residues are the general acid and base; and (ii) an aspartic acid residue plays a role of recognizing adenine moiety of the substrate.
