Structural and functional studies of cinnamomin, a new type II ribosome-inactivating protein isolated from the seeds of the camphor tree.
Cinnamomin is a new type II ribosome-inactivating protein (RIP). Its A-chain exhibits RNA N-glycosidase activity to inactivate the ribosome and thus inhibit protein synthesis, whereas the glycosylated B-chain is a lectin. The primary structure of cinnamomin, which exhibits approximately 55% identity with those of ricin and abrin, was deduced from the nucleotide sequences of cDNAs of cinnamomin A- and B-chains. It is composed of a total of 549 amino-acid residues: 271 residues in the A-chain, a 14-residue linker and 264 residues in the B-chain. To explore its biological function, the cinnamomin A-chain was expressed in Escherichia coli with a yield of 100 mg per L of culture, and purified through two-step column chromatography. After renaturation, the recovery of the enzyme activity of the expressed A-chain was 80% of that of native A-chain. Based on the modeling of the three-dimensional structure of the A-chain, the functional roles of five amino acids and the only cysteine residues were investigated by site-directed mutagenesis or chemical modification. The conserved single mutation of the five amino-acid residues led to 8-50-fold losses of enzymatic activity, suggesting that these residues were crucial for maintaining the RNA N-glycosidase activity of the A-chain. Most interestingly, the strong electric charge introduced at the position of the single cysteine in A-chain seemed to play a role in enzyme/substrate binding.
Xie
L
L
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Wang
B Z
BZ
Hu
R G
RG
Ji
H B
HB
Zhang
L
L
Liu
W Y
WY
D020418:Algal Proteins; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D054790:Ribosome Inactivating Proteins, Type 2; D012270:Ribosomes; D012639:Seeds / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014197:Trees / Q000196:embryology*
D020418:Algal Proteins; D018076:DNA, Complementary; D010940:Plant Proteins; D011506:Proteins; D054790:Ribosome Inactivating Proteins, Type 2; C061930:cinnamomin, Phytophthora cinnamomi