1a47: Difference between revisions

Revision as of 09:47, 2 May 2008

CGTASE FROM THERMOANAEROBACTERIUM THERMOSULFURIGENES EM1 IN COMPLEX WITH A MALTOHEXAOSE INHIBITOR

OverviewOverview

The product specificity and pH optimum of the thermostable cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes EM1 was engineered using a combination of x-ray crystallography and site-directed mutagenesis. Previously, a crystal soaking experiment with the Bacillus circulans strain 251 beta-CGTase had revealed a maltononaose inhibitor bound to the enzyme in an extended conformation. An identical experiment with the CGTase from T. thermosulfurigenes EM1 resulted in a 2.6-A resolution x-ray structure of a complex with a maltohexaose inhibitor, bound in a different conformation. We hypothesize that the new maltohexaose conformation is related to the enhanced alpha-cyclodextrin production of the CGTase. The detailed structural information subsequently allowed engineering of the cyclodextrin product specificity of the CGTase from T. thermosulfurigenes EM1 by site-directed mutagenesis. Mutation D371R was aimed at hindering the maltohexaose conformation and resulted in enhanced production of larger size cyclodextrins (beta- and gamma-CD). Mutation D197H was aimed at stabilization of the new maltohexaose conformation and resulted in increased production of alpha-CD. Glu258 is involved in catalysis in CGTases as well as alpha-amylases, and is the proton donor in the first step of the cyclization reaction. Amino acids close to Glu258 in the CGTase from T. thermosulfurigenes EM1 were changed. Phe284 was replaced by Lys and Asn327 by Asp. The mutants showed changes in both the high and low pH slopes of the optimum curve for cyclization and hydrolysis when compared with the wild-type enzyme. This suggests that the pH optimum curve of CGTase is determined only by residue Glu258.

About this StructureAbout this Structure

1A47 is a Single protein structure of sequence from Thermoanaerobacterium thermosulfurigenes. Full crystallographic information is available from OCA.

ReferenceReference

Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1., Wind RD, Uitdehaag JC, Buitelaar RM, Dijkstra BW, Dijkhuizen L, J Biol Chem. 1998 Mar 6;273(10):5771-9. PMID:9488711 Page seeded by OCA on Fri May 2 09:47:14 2008

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OCA

@@ Line 1: / Line 1: @@
 [[Image:1a47.gif|left|200px]]
- {{Structure
+<!--
-|PDB= 1a47 |SIZE=350|CAPTION= <scene name='initialview01'>1a47</scene>, resolution 2.56&Aring;
+The line below this paragraph, containing "STRUCTURE_1a47", creates the "Structure Box" on the page.
-|SITE= <scene name='pdbsite=AM1:Sugar+Binding+Subsite+-1+In+The+Active+Site'>AM1</scene>, <scene name='pdbsite=AM2:Sugar+Binding+Subsite+-2+In+The+Active+Site'>AM2</scene>, <scene name='pdbsite=AM3:Sugar+Binding+Subsite+-3+In+The+Active+Site'>AM3</scene>, <scene name='pdbsite=AP1:Sugar+Binding+Subsite++1+In+The+Active+Site+(Catalytic+Site)'>AP1</scene>, <scene name='pdbsite=AP2:Sugar+Binding+Subsite++2+In+The+Active+Site'>AP2</scene>, <scene name='pdbsite=AP3:Sugar+Binding+Subsite++3+In+The+Active+Site'>AP3</scene>, <scene name='pdbsite=BS3:Sugar+Binding+Site+3+(Homologous+To+Mbs3+In+Pdb+Entry+1cdg)'>BS3</scene>, <scene name='pdbsite=CA1:Ca+Binding+Site'>CA1</scene> and <scene name='pdbsite=CA2:Ca+Binding+Site'>CA2</scene>
+You may change the PDB parameter (which sets the PDB file loaded into the applet)
-|LIGAND= <scene name='pdbligand=ACI:6-AMINO-4-HYDROXYMETHYL-CYCLOHEX-4-ENE-1,2,3-TRIOL'>ACI</scene>, <scene name='pdbligand=CA:CALCIUM+ION'>CA</scene>, <scene name='pdbligand=G6D:6-DEOXY-ALPHA-D-GLUCOSE'>G6D</scene>, <scene name='pdbligand=GLC:GLUCOSE'>GLC</scene>, <scene name='pdbligand=GTE:GLUCOSE+4-O4+GROUP'>GTE</scene>
+or the SCENE parameter (which sets the initial scene displayed when the page is loaded),
-|ACTIVITY= <span class='plainlinks'>[http://en.wikipedia.org/wiki/Cyclomaltodextrin_glucanotransferase Cyclomaltodextrin glucanotransferase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.4.1.19 2.4.1.19] </span>
+or leave the SCENE parameter empty for the default display.
-|GENE= AMYA ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=33950 Thermoanaerobacterium thermosulfurigenes])
+-->
-|DOMAIN=
+{{STRUCTURE_1a47|  PDB=1a47  |  SCENE=  }}
-|RELATEDENTRY=
-|RESOURCES=<span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=1a47 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1a47 OCA], [http://www.ebi.ac.uk/pdbsum/1a47 PDBsum], [http://www.rcsb.org/pdb/explore.do?structureId=1a47 RCSB]</span>
-}}
 '''CGTASE FROM THERMOANAEROBACTERIUM THERMOSULFURIGENES EM1 IN COMPLEX WITH A MALTOHEXAOSE INHIBITOR'''
@@ Line 30: / Line 27: @@
 [[Category: Rozeboom, H J.]]
 [[Category: Uitdehaag, J C.M.]]
-[[Category: acarbose]]
+[[Category: Acarbose]]
-[[Category: family 13 glycosyl hydrolase]]
+[[Category: Family 13 glycosyl hydrolase]]
-[[Category: glycosidase]]
+[[Category: Glycosidase]]
-[[Category: ligand]]
+[[Category: Ligand]]
-[[Category: substrate]]
+[[Category: Substrate]]
-[[Category: thermostable]]
+[[Category: Thermostable]]
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May  2 09:47:14 2008''
-''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun Mar 30 18:32:51 2008''