Crystal structures of the ribonuclease MC1 from bitter gourd seeds, complexed with 2'-UMP or 3'-UMP, reveal structural basis for uridine specificity.
Ribonuclease MC1 (RNase MC1) isolated from seeds of bitter gourd (Momordica charantia) consists of 190 amino acids and is characterized by a preferential cleavage at the 5'-side of uridine. This uridine specificity distinguishes RNase MC1 from other enzymes belonging to the RNase T2 family. The three-dimensional structures of RNase MC1, in a complex with either 2'-UMP or 3'-UMP, were determined at 1.48 and 1.77 A resolutions, respectively. The side chains of Gln9 and Asn71 interact with O4 and N3, respectively, of the uracil base by hydrogen bondings. In addition, the uracil base is sandwiched by the hydrophobic side chains of Leu73 and Phe80. Compared with these amino acid residues and corresponding residues in RNases in the RNase T2 family, Gln9 and Phe80 are highly conserved in the RNases in T2 family, while Asn71 and Leu73 in RNase MC1 are variant in sequences. It is thus likely that interactions of the side chains of Asn71 and Leu73 with the uracil base are responsible for the absolute uridine specificity of RNase MC1. Site-directed mutagenesis experiments showed that replacement of Asn by Thr decreased both the catalytic efficiency and the binding affinity by 2.3- and 7.0-fold, respectively, and substitution of Leu73 for Ala predominantly decreased the binding affinity by 14. 5-fold, compared with findings in case of wild-type RNase MC1. It is thus demonstrated that Asn71 and Leu73 play an essential role in uridine preference for RNase MC1.
Suzuki
A
A
Division of Biological Sciences, Hokkaido University, Sapporo, 060-0810, Japan.
Yao
M
M
Tanaka
I
I
Numata
T
T
Kikukawa
S
S
Yamasaki
N
N
Kimura
M
M
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014529:Uridine / Q000378:metabolism*; D014542:Uridine Monophosphate / Q000737:chemistry / Q000378:metabolism*
D014542:Uridine Monophosphate; D012260:Ribonucleases; C413748:ribonuclease MC1; D014529:Uridine