Expression and mutational analysis of amino acid residues involved in catalytic activity in a ribonuclease MC1 from the seeds of bitter gourd.
The ribonuclease MC1 (RNase MC1) from seeds of bitter gourd (Momordica charantia) consists of 190 amino acids and belongs to the RNase T2 family, including fungal RNases typified by RNase Rh from Rhizopus niveus. We expressed RNase MC1 in Escherichia coli cells and made use of site-directed mutagenesis to identify essential amino acid residues for catalytic activity. Mutations of His34 and His88 to Ala completely abolished the enzymatic activity, and considerable decreases in the enzymatic activity were observed in cases of mutations of His83, Glu84, and Lys87, when yeast RNA was used as a substrate. Kinetic parameters for the enzymatic activity of the mutants of His83, Glu84, and Lys87 were analyzed using a dinucleoside monophosphate CpU. Km values for the mutants were approximately like that for wild-type, while k(cat) values were decreased by about 6 to 25-fold. These results suggest that His34, His83, Glu84, Lys87, and His88 in RNase MC1 may be involved in the catalytic function. These observation suggests that RNase MC1 from a plant catalyzes RNA degradation in a similar manner to that of fungal RNases.
Numata
T
T
Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
Kashiba
T
T
Hino
M
M
Funatsu
G
G
Ishiguro
M
M
Yamasaki
N
N
Kimura
M
M
D000596:Amino Acids; D020134:Catalytic Domain; D019662:Cucurbitaceae / Q000201:enzymology*; D004722:Endoribonucleases / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000201:enzymology
D000596:Amino Acids; D011993:Recombinant Fusion Proteins; D004722:Endoribonucleases; C022543:ribonuclease T(2)