adenine-bound MTAN1 is exclusively in its closed form, substantial conformational changes occur in this region during the enzymatic reaction. Residues Met35, Val90, Leu181, Met201, Phe237 and Ile143', Ile145' and Phe148' in a neighboring MTAN1 monomer bind adenine via hydrophobic and van der Waals interactions. The hydrophobic side chains Phe237 and Leu181 show structural perturbations that provide the flexible accommodation of the adenine ring. The carboxylic side chain of Asp225 hydrogen bonds with nitrogen atoms in the adenine ring, therefore being key determinant for the adenine base. The side chain Thr116 and backbone Gly118 also interact with the adenine molecule
hydrolysis of 5'-methylthioadenosine predominates despite MTAN2 possessing both 5'-methylthioadenosine and S-adenosylhomocysteine hydrolysis activity. MTAN2 binds 5'-methylthioadenosine approximately 6fold higher than S-adenosylhomocysteine. Residues between 216 and 225 in MTAN2 contain conformational differences representing an open conformation when compared with MTAN1. The side chains Met22, Val78, Met168, Met188, Phe224 along with Ile130', Val132' and Leu135' in a neighboring MTAN2 monomer bind adenine via hydrophobic and van der Waals interactions. The hydrophobic side chains Phe224 and Met168 show structural perturbations that provide the flexible accommodation of the adenine ring. The carboxylic side chain of Asp211 hydrogen bonds with nitrogen atoms in the adenine ring, therefore being key determinant for the adenine base. The side chain Thr103 and backbone Gly105 also interact with the adenine molecule
the plant active site is likely less flexible, which may contribute to its reduced efficiency in binding larger substrates. The 5'-alkylthio binding subsite is more constricted in MTAN1
the plant active site is likely less flexible, which may contribute to its reduced efficiency in binding larger substrates. The 5'-alkylthio binding subsite is more constricted in MTAN1
the plant active site is likely less flexible, which may contribute to its reduced efficiency in binding larger substrates. The 5'-alkylthio binding subsite is more constricted in MTAN1
combination of a more occluded active site in its open state and reduced ligand-induced conformational changes in the plant in contrast to Escherichia coli MTAN
inhibits only in the presence of Ca2+, MTAN interacts specifically with calcineurin B-like 3, but not with calcineurin B-like 1 and calcineurin B-like 4. The 91-amino acid region of calcineurin B-like 3 is sufficient for the interaction with MTAN. Calcineurin B-like 3 and MTAN associate with each other in plant cells, they interact in vitro and in vivo, form a complex outside of the nucleus, colocalize mainly at the plasma membrane
localized at the plasma membrane, in the cytoplasm and in the nucleus of transgenic tobacco plants. Abundant in the membrane structure and weak in the cytoplasm of onion epidermal cells
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
MTAN1 in its apo form and in complex with formycin A and 5'-methylthiotubercidin to a resolution of 2.0 A, 1.9 A and 1.8 A, respectively. By hanging-drop, vapor-diffusion technique. Belongs to space group P21. The monomer of MTAN1 has a mixed alpha/beta structure composed of a twisted, ten-stranded mixed beta sheet flanked by seven alpha helices and two short 3(10) helices. In the MTAN1 crystals, the asymmetric unit contains two monomers, which interact via an interface involving the four loops beta2-beta3, beta4-alpha2, beta6-alpha2b and beta8-alpha4, and the alpha2, alpha2b, and alpha5 helices. Both polar and apolar residues are involved in hydrogen bond and van der Waals interactions
in complex with adenine, by sitting- and hanging-drop formation at 22°C, at 2.9 A resolution. Belongs to space group P3121, monomer consists of seven alpha-helices, ten beta-strands, and a 3(10)-helix. Residues between 216 and 225 demonstrate weak electron density for both subunits, therefore indicating this loop has high flexibility. Bound adenine is located in the deep pocket formed by the monomer with the entrance partially covered by the adjacent subunit. This flap is critical in the formation of a wide dimer interface
can reduce steric hindrance at the end of the 5'-alkylthio binding subsite, so that longer 5'-substituents may be accommodated more easily in the MTAN2 active site
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
different regions of MTAN cloned into the pGBT9.BS vector and transformed into Y190 yeast cells carrying pGAD.GH or pGAD.CBL3. Expressed in Escherichia coli BL21 cells carrying a GST fusion construct. MTAN promoter-GUS construct (pBI.tMTAN) transformed into Agrobacterium tumefaciens strain GV3101 and introduced into Arabidopsis plants by the floral dip method. 35S::CFP-CBL3-Myc and 35S::YFP-MTANHA constructs introduced, either alone or in combination, into Nicotiana benthamiana leaves by Agrobacterium tumefaciens-mediated infiltration method. CBL3-GFP and MTAN-GFP chimeric genes under the control of the cauliflower mosaic virus 35S promoter (pMD.CBL3 and pMD.MTAN) transiently expressed in Allium cepa epidermal cells. Onion epidermal cells bombarded with fusion constructs CBL3-YN and MTAN-YC
into the pET28a+ vector at the NdeI and HindIII sites, overexpression of the enzyme with a cleavable N-terminal His6 tag in Escherichia coli BL21 Codon+ cells
into the pET28a+ vector at the NdeI and HindIII sites, overexpression of the enzyme with a cleavable N-terminal His6 tag in Escherichia coli BL21 Codon+ cells