2e8z: Difference between revisions

==Overview==

The AmyX gene encoding pullulanase from the common spore-forming bacterium Bacillus subtilis strain 168 was cloned, overexpressed in Escherichia coli, purified and crystallized. The recombinant pullulanase was purified to homogeneity using ammonium sulfate precipitation, hydrophobic chromatography and anion-exchange chromatography, resulting in a specific activity of 24.10 U per milligram of protein. SDS-PAGE analysis showed that the molecular weight of the protein is approximately 81.0 kDa, which is similar to the calculated molecular weight, 81.1 kDa, from its translated cDNA sequence. The k(cat) and K(m) of the purified enzyme with pullulan as substrate were approximately 79 s(-1) and 1.284 mg ml(-1), respectively. X-ray crystallographic analysis of the pullulanase crystal showed that the crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 70.568, b = 127.68, c = 189.25 angstroms. The crystal contains two molecules of pullulanase in the asymmetric unit, with a solvent content of 53.15%. The crystal diffracted to 2.1 angstroms resolution at a synchrotron and is suitable for structure determination.

==About this Structure==

2E8Z is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bacillus_subtilis Bacillus subtilis] with <scene name='pdbligand=CA:'>CA</scene>, <scene name='pdbligand=ACT:'>ACT</scene> and <scene name='pdbligand=GOL:'>GOL</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Pullulanase Pullulanase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.41 3.2.1.41] Known structural/functional Sites: <scene name='pdbsite=AC1:Glc+Binding+Site+For+Residue+A+721'>AC1</scene>, <scene name='pdbsite=AC2:Glc+Binding+Site+For+Residue+A+722'>AC2</scene>, <scene name='pdbsite=AC3:Glc+Binding+Site+For+Residue+A+723'>AC3</scene>, <scene name='pdbsite=AC4:Glc+Binding+Site+For+Residue+A+724'>AC4</scene>, <scene name='pdbsite=AC5:Glc+Binding+Site+For+Residue+A+725'>AC5</scene>, <scene name='pdbsite=AC6:Glc+Binding+Site+For+Residue+A+726'>AC6</scene>, <scene name='pdbsite=AC7:Glc+Binding+Site+For+Residue+B+731'>AC7</scene>, <scene name='pdbsite=AC8:Glc+Binding+Site+For+Residue+B+732'>AC8</scene>, <scene name='pdbsite=AC9:Glc+Binding+Site+For+Residue+B+733'>AC9</scene>, <scene name='pdbsite=BC1:Glc+Binding+Site+For+Residue+B+734'>BC1</scene>, <scene name='pdbsite=BC2:Glc+Binding+Site+For+Residue+B+735'>BC2</scene>, <scene name='pdbsite=BC3:Glc+Binding+Site+For+Residue+B+736'>BC3</scene>, <scene name='pdbsite=BC4:Ca+Binding+Site+For+Residue+A+741'>BC4</scene>, <scene name='pdbsite=BC5:Ca+Binding+Site+For+Residue+B+742'>BC5</scene>, <scene name='pdbsite=BC6:Act+Binding+Site+For+Residue+A+751'>BC6</scene>, <scene name='pdbsite=BC7:Act+Binding+Site+For+Residue+B+752'>BC7</scene>, <scene name='pdbsite=BC8:Gol+Binding+Site+For+Residue+A+761'>BC8</scene> and <scene name='pdbsite=BC9:Gol+Binding+Site+For+Residue+B+762'>BC9</scene>. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2E8Z OCA].

 

[[Category: Pullulanase]]

[[Category: Single protein]]

[[Category: Iwamoto, H.]]

[[Category: Katsuya, Y.]]

[[Category: Malle, D.]]

[[Category: Mikami, B.]]

[[Category: Utsumi, S.]]

[[Category: beta-alpha-barrel]]