• BMB Reports
• /
• v.30 no.6
• /
• pp.410-414
• /
• 1997

We purified a protein carboxyl O-methyltransferase (protein methylase II) from porcine spleen to homogeneity. The molecular weight of the porcine spleen protein methylase II (ps-PM II) was estimated to be 27,500 daltons on SDS-PAGE. Amino acid sequence of N-terminal 28 residues for ps-PM II was identified. Amino-terminal three amino acid residues of ps-PM II were deleted when compared to those of other protein carboxyl methytransferase. S-Adenosyl-L-homocysteine competitively inhibits ps-PM II with a K, value of $1.63{\times}10^{-7}M$. Myelin basic protein exhibited the highest methyl-accepting capacity among the proteins tested.

• YAKHAK HOEJI
• /
• v.35 no.6
• /
• pp.473-482
• /
• 1991

Protein methylase II (S-adenosyl-L-methionine:protein carboxyl-O-methyltransferase; EC 2.1.1.24., PM II) was purified from chicken pancreas by subcellular fractionation, DEAE-cellulose chromatography, QAE-Sephadex A-50 chromatography, Sephadex G-75 chromatography, and Sephadex G-75 rechromatography. The purified PM II gave a single band upon polyarcrylamide gel electrophoresis both in the presence of SDS and in Tris glycine buffer without SDS. The pI value of purified PM II was identified as 5.7 on isoelectric focusing gel. Properties and activities of PM II were studied and the following results were obtained. 1) PM II from chicken pancreas was purified approximately 221-fold with a yield of 1.3%. 2) The purified PM II appear constituted of a single polypeptide chain of a molecular weight 46,800 daltons. 3) Hemoglobin exhibited the highest of methyl-accepting activity among the substrates tested. 4) The purified PM II has a $K_m$ of $4.67{\times}10^{-6}M$ and a $V_{max}$ of 37.5 pmoles of $methyl-^{14}C/min./mg$ enzyme for $SAM^{-14}CH_3$ as methyl donor in the presence of histone type II-As. 5) It is found that S-adenosyl-L-homocysteine is a competitive inhibitor for PM II with $K_i$ value of $3.23{\times}10^{-5}M$.

• Bulletin of the Korean Chemical Society
• /
• v.28 no.6
• /
• pp.941-946
• /
• 2007

Bacillus halodurans O-methyltransferase (BhOMT) is a S-adenosylmethionine (SAM or AdoMet) dependent methyltransferase. Three dimensional structure of the BhOMT bound to S-adenosyl-L-homocysteine (SAH or AdoHcy) has been determined by comparative homology modeling. BhOMT has 40% sequence identity with caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) from alfalfa. Based on x-ray structure of CCoAOMT, three dimensional structure of BhOMT was determined using MODELLER. The substrate binding sites of these two proteins showed slight differences, but these differences were important to characterize the substrate of BhOMT. Automated docking study showed that four flavonoids, quercetin, fisetin, myricetin, and luteolin which have two hydroxyl groups simultaneously at 3'- and 4'-position in the B-ring and structural rigidity of Cring resulting from the double bond characters between C2 and C3, were well docked as ligands of BhOMT. These flavonoids form stable hydrogen bondings with K211, R170, and hydroxyl group at 3'-position in the Bring has stable electrostatic interaction with Ca2+ ion in BhOMT. This study will be helpful to understand the biochemical function of BhOMT as an O-methyltransferase for flavonoids.

• BMB Reports
• /
• v.34 no.6
• /
• pp.559-565
• /
• 2001

Protein carboxyl O-methyltransferase (E.C.2.1.1.24) may play a role in the repair of aged protein that is spontaneously incorporated with isoaspartyl residues. The porcine brain carboxyl O-methyltransferase was cloned in the pET32 vector, and overexpressed in E.coh (BL21) that harbors pETPCMT, which encodes 227 amino acids, including tagging proteins at the N-terminus. The protein sequence of the cloned porcine brain PCMT (r-pbPCMT) shares a 98% identity with that of human erythrocyte PCMT and rat brain PCMT. It is 100% identical with that of bovine brain. The r-pbPCMT was purified using Ni-NTA affinity chromatography and digested by enterokinase in order to remove the protein tags. Then Superdex 75HR gel filtration chromatography was performed. The r-pbPCMT exhibited similar in vitro substrate specificities with the PCMT that was purified from porcine brain. The molecular weight of the enzyme was estimated to be 24.5 kDa on the SDS polyacrylamide gel electrophoresis. The $K_m$ value was $1.1{\times}10^{-7}\;M$ for S-adenosyl-L-methionine. S-adnosyl-L-homocysteine was a competitive type of inhibitor with the $K_i$ value of $1.38{\times}10^{-4}\;M$. The enzyme has optimal activity at pH 6.0 and $37^{\circ}C$. These results indicate that the expressed enzyme is functionally similar to the natural protein. It also suggests that it may be a suitable model to further understand the function of the mammalian enzyme.

• The Plant Pathology Journal
• /
• v.32 no.5
• /
• pp.377-387
• /
• 2016

Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus, is one of the most important viruses of cultivated tomatoes worldwide, mainly causing yellowing and curling of leaves with stunting in plants. TYLCV causes severe problems in sub-tropical and tropical countries, as well as in Korea. However, the mechanism of TYLCV infection remains unclear, although the function of each viral component has been identified. TYLCV C4 codes for a small protein involved in various cellular functions, including symptom determination, gene silencing, viral movement, and induction of the plant defense response. In this study, through yeast-two hybrid screenings, we identified TYLCV C4-interacting host proteins from both healthy and symptom-exhibiting tomato tissues, to determine the role of TYLCV C4 proteins in the infection processes. Comparative analyses of 28 proteins from healthy tissues and 36 from infected tissues showing interactions with TYLCV C4 indicated that TYLCV C4 mainly interacts with host proteins involved in translation, ubiquitination, and plant defense, and most interacting proteins differed between the two tissues but belong to similar molecular functional categories. Four proteins-two ribosomal proteins, S-adenosyl-L-homocysteine hydrolase, and 14-3-3 family protein-were detected in both tissues. Furthermore, the identified proteins in symptom-exhibiting tissues showed greater involvement in plant defenses. Some are key regulators, such as receptor-like kinases and pathogenesis-related proteins, of plant defenses. Thus, TYLCV C4 may contribute to the suppression of host defense during TYLCV infection and be involved in ubiquitination for viral infection.