1dtu: Difference between revisions

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BACILLUS CIRCULANS STRAIN 251 CYCLODEXTRIN GLYCOSYLTRANSFERASE: A MUTANT Y89D/S146P COMPLEXED TO AN HEXASACCHARIDE INHIBITOR

OverviewOverview

Cyclodextrin glycosyltransferases (CGTase) (EC 2.4.1.19) are extracellular bacterial enzymes that generate cyclodextrins from starch. All known CGTases produce mixtures of alpha, beta, and gamma-cyclodextrins. A maltononaose inhibitor bound to the active site of the CGTase from Bacillus circulans strain 251 revealed sugar binding subsites, distant from the catalytic residues, which have been proposed to be involved in the cyclodextrin size specificity of these enzymes. To probe the importance of these distant substrate binding subsites for the alpha, beta, and gamma-cyclodextrin product ratios of the various CGTases, we have constructed three single and one double mutant, Y89G, Y89D, S146P and Y89D/S146P, using site-directed mutagenesis. The mutations affected the cyclization, coupling; disproportionation and hydrolyzing reactions of the enzyme. The double mutant Y89D/S146P showed a twofold increase in the production of alpha-cyclodextrin from starch. This mutant protein was crystallized and its X-ray structure, in a complex with a maltohexaose inhibitor, was determined at 2.4 A resolution. The bound maltohexaose molecule displayed a binding different from the maltononaose inhibitor, allowing rationalization of the observed change in product specificity. Hydrogen bonds (S146) and hydrophobic contacts (Y89) appear to contribute strongly to the size of cyclodextrin products formed and thus to CGTase product specificity. Changes in sugar binding subsites -3 and -7 thus result in mutant proteins with changed cyclodextrin production specificity.

About this StructureAbout this Structure

1DTU is a Single protein structure of sequence from Bacillus circulans with and as ligands. Active as Cyclomaltodextrin glucanotransferase, with EC number 2.4.1.19 Full crystallographic information is available from OCA.

ReferenceReference

Rational design of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 to increase alpha-cyclodextrin production., van der Veen BA, Uitdehaag JC, Penninga D, van Alebeek GJ, Smith LM, Dijkstra BW, Dijkhuizen L, J Mol Biol. 2000 Mar 3;296(4):1027-38. PMID:10686101

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-[[Image:1dtu.jpg|left|200px]]<br /><applet load="1dtu" size="450" color="white" frame="true" align="right" spinBox="true"
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 caption="1dtu, resolution 2.40&Aring;" />
 '''BACILLUS CIRCULANS STRAIN 251 CYCLODEXTRIN GLYCOSYLTRANSFERASE: A MUTANT Y89D/S146P COMPLEXED TO AN HEXASACCHARIDE INHIBITOR'''<br />
 ==Overview==
-Cyclodextrin glycosyltransferases (CGTase) (EC 2.4.1.19) are extracellular, bacterial enzymes that generate cyclodextrins from starch. All known, CGTases produce mixtures of alpha, beta, and gamma-cyclodextrins. A, maltononaose inhibitor bound to the active site of the CGTase from, Bacillus circulans strain 251 revealed sugar binding subsites, distant, from the catalytic residues, which have been proposed to be involved in, the cyclodextrin size specificity of these enzymes. To probe the, importance of these distant substrate binding subsites for the alpha, beta, and gamma-cyclodextrin product ratios of the various CGTases, we, have constructed three single and one double mutant, Y89G, Y89D, S146P and, Y89D/S146P, using site-directed mutagenesis. The mutations affected the, cyclization, coupling; disproportionation and hydrolyzing reactions of the, enzyme. The double mutant Y89D/S146P showed a twofold increase in the, production of alpha-cyclodextrin from starch. This mutant protein was, crystallized and its X-ray structure, in a complex with a maltohexaose, inhibitor, was determined at 2.4 A resolution. The bound maltohexaose, molecule displayed a binding different from the maltononaose inhibitor, allowing rationalization of the observed change in product specificity., Hydrogen bonds (S146) and hydrophobic contacts (Y89) appear to contribute, strongly to the size of cyclodextrin products formed and thus to CGTase, product specificity. Changes in sugar binding subsites -3 and -7 thus, result in mutant proteins with changed cyclodextrin production, specificity.
+Cyclodextrin glycosyltransferases (CGTase) (EC 2.4.1.19) are extracellular bacterial enzymes that generate cyclodextrins from starch. All known CGTases produce mixtures of alpha, beta, and gamma-cyclodextrins. A maltononaose inhibitor bound to the active site of the CGTase from Bacillus circulans strain 251 revealed sugar binding subsites, distant from the catalytic residues, which have been proposed to be involved in the cyclodextrin size specificity of these enzymes. To probe the importance of these distant substrate binding subsites for the alpha, beta, and gamma-cyclodextrin product ratios of the various CGTases, we have constructed three single and one double mutant, Y89G, Y89D, S146P and Y89D/S146P, using site-directed mutagenesis. The mutations affected the cyclization, coupling; disproportionation and hydrolyzing reactions of the enzyme. The double mutant Y89D/S146P showed a twofold increase in the production of alpha-cyclodextrin from starch. This mutant protein was crystallized and its X-ray structure, in a complex with a maltohexaose inhibitor, was determined at 2.4 A resolution. The bound maltohexaose molecule displayed a binding different from the maltononaose inhibitor, allowing rationalization of the observed change in product specificity. Hydrogen bonds (S146) and hydrophobic contacts (Y89) appear to contribute strongly to the size of cyclodextrin products formed and thus to CGTase product specificity. Changes in sugar binding subsites -3 and -7 thus result in mutant proteins with changed cyclodextrin production specificity.
 ==About this Structure==
-DTU is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bacillus_circulans Bacillus circulans] with CA and ADH as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [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] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1DTU OCA].
+DTU is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bacillus_circulans Bacillus circulans] with <scene name='pdbligand=CA:'>CA</scene> and <scene name='pdbligand=ADH:'>ADH</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [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] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1DTU OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Cyclomaltodextrin glucanotransferase]]
 [[Category: Single protein]]
-[[Category: Dijkstra, B.W.]]
+[[Category: Dijkstra, B W.]]
-[[Category: Kalk, K.H.]]
+[[Category: Kalk, K H.]]
-[[Category: Uitdehaag, J.C.M.]]
+[[Category: Uitdehaag, J C.M.]]
 [[Category: ADH]]
 [[Category: CA]]
@@ Line 27: / Line 27: @@
 [[Category: product specificity]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 13:34:28 2007''
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