Pokeweed antiviral protein region Gly209-Lys225 is critical for RNA N-glycosidase activity of the prokaryotic ribosome.
Pokeweed antiviral protein (PAP) isolated from Phytolacca americana is a ribosome-inactivating protein (RIP) that has RNA N-glycosidase (RNG) activity towards both eukaryotic and prokaryotic ribosomes. In contrast, karasurin-A (KRN), a RIP from Trichosanthes kirilowii var. japonica, is active only on eukaryotic ribosomes. Stepwise selection of chimera proteins between PAP and KRN indicated that the C-terminal region of PAP (residues 209-225) was critical for RNG activity toward prokaryotic ribosomes. When the region of PAP (residues 209-225) was replaced with the corresponding region of KRN the PAP chimera protein, like KRN, was active only on eukaryotic ribosomes. Furthermore, insertion of the region of PAP (residues 209-225) into the KRN chimera protein resulted not only in the detectable RNG activity toward prokaryotic ribosome, but also activity toward the eukaryotic ribosomes as well that was seven-fold higher than for the original KRN. In this study, the possibility of genetic manipulation of the activity and substrate specificity of RIPs is demonstrated.
Nagasawa
Yoshimi
Y
School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
Fujii
Kazuyuki
K
Yoshikawa
Takafumi
T
Kobayashi
Yoshinori
Y
Kondo
Toshiya
T
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D011387:Prokaryotic Cells / Q000502:physiology*; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D054788:Ribosome Inactivating Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D054789:Ribosome Inactivating Proteins, Type 1 / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D012270:Ribosomes / Q000502:physiology*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C069870:karasurin protein, Trichosanthes kirilowii; D012313:RNA; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins; C024275:pokeweed antiviral protein