1UCG

Crystal structure of Ribonuclease MC1 N71T mutant

Summary for 1UCG

• Entry DOI: 10.2210/pdb1ucg/pdb
• Related: 1BK7 1J1F 1J1G 1UCA 1UCC 1UCD
• Descriptor: Ribonuclease MC, MANGANESE (II) ION (3 entities in total)
• Functional Keywords: alpha plus beta, hydrolase
• Biological source: Momordica charantia (balsam pear)
• Total number of polymer chains: 2
• Total formula weight: 42615.82

Authors

Suzuki, A.,Numata, T.,Yao, M.,Tanaka, I.,Kimura, M. (deposition date: 2003-04-14, release date: 2003-04-29, Last modification date: 2023-10-25)

Primary citation

Numata, T.,Suzuki, A.,Kakuta, Y.,Kimura, K.,Yao, M.,Tanaka, I.,Yoshida, Y.,Ueda, T.,Kimura, M.
Crystal structures of the ribonuclease MC1 mutants N71T and N71S in complex with 5'-GMP: structural basis for alterations in substrate specificity
Biochemistry, 42:5270-5278, 2003

PubMed Abstract: Ribonuclease MC1 (RNase MC1), isolated from bitter gourd seeds, is a uridine specific RNase belonging to the RNase T2 family. Mutations of Asn71 in RNase MC1 to the amino acids Thr (N71T) and Ser (N71S) in guanosine preferential RNases altered the substrate specificity from uridine specific to guanosine specific, as shown by the transphosphorylation of diribonucleoside monophosphates [Numata, T., et al. (2001) Biochemistry 40, 524-530]. To elucidate the structural basis for the alteration of substrate specificity, crystal structures of the RNase MC1 mutants N71T and N71S, free or complexed with 5'-GMP, were determined at resolutions higher than 2 A. In the N71T-5'-GMP and N71S-5'-GMP complexes, the guanine moiety was, as in the case of the uracil moiety bound to wild-type RNase MC1, firmly stabilized in the B2 site by an extensive network of hydrogen bonds and hydrophobic interactions. Structure comparisons showed that mutations of Asn71 to Thr or Ser cause an enlargement of the B2 site, which then make it feasible to insert a guanine base into the B2 site of mutants N71T and N71S. This binding further allows for hydrogen bonding interaction of the side chain hydroxyl groups of Thr71 or Ser71 with the N7 atom of the guanine base. The mode of guanine binding of mutants N71T and N71S was found to be essentially identical to that of a guanosine preferential RNase NW from Nicotiana glutinosa. In particular, hydrogen bonds between the N7 atom of the guanine base and the hydroxyl groups of the amino acids at position 71 (RNase MC1 numbering) were completely conserved in three guanosine preferential enzymes, thereby indicating that the hydrogen bond may play an essential role in guanine binding in guanosine preferential RNases in the RNase T2 family. Consequently, it can be concluded that amino acids at position 71 (RNase MC1 numbering) serve as one of the determinants for substrate specificity (or preference) in the RNase T2 fimily by changing the size and shape of the B2 site.

PubMed: 12731868
DOI: 10.1021/bi034103g

Experimental method

X-RAY DIFFRACTION (1.65 Å)