1q2e: Difference between revisions

Revision as of 15:35, 21 February 2008

CELLOBIOHYDROLASE CEL7A WITH LOOP DELETION 245-252 AND BOUND NON-HYDROLYSABLE CELLOTETRAOSE

OverviewOverview

The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations.The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K(M) and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3)+Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.

About this StructureAbout this Structure

1Q2E is a Single protein structure of sequence from Hypocrea jecorina with and as ligands. Active as Cellulose 1,4-beta-cellobiosidase, with EC number 3.2.1.91 Full crystallographic information is available from OCA.

ReferenceReference

Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D., von Ossowski I, Stahlberg J, Koivula A, Piens K, Becker D, Boer H, Harle R, Harris M, Divne C, Mahdi S, Zhao Y, Driguez H, Claeyssens M, Sinnott ML, Teeri TT, J Mol Biol. 2003 Oct 31;333(4):817-29. PMID:14568538

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-[[Image:1q2e.jpg|left|200px]]<br /><applet load="1q2e" size="450" color="white" frame="true" align="right" spinBox="true"
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 caption="1q2e, resolution 1.75&Aring;" />
 '''CELLOBIOHYDROLASE CEL7A WITH LOOP DELETION 245-252 AND BOUND NON-HYDROLYSABLE CELLOTETRAOSE'''<br />
 ==Overview==
-The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof, of the active site tunnel at the catalytic centre. Mutants were designed, to study the role of this loop in crystalline cellulose degradation. A, hydrogen bond to substrate made by a tyrosine at the tip of the loop was, removed by the Y247F mutation. The mobility of the loop was reduced by, introducing a new disulphide bridge in the mutant D241C/D249C. The tip of, the loop was deleted in mutant Delta(G245-Y252). No major structural, disturbances were observed in the mutant enzymes, nor was the, thermostability of the enzyme affected by the mutations.The Y247F mutation, caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a, small effect on cellulose hydrolysis. Deletion of the tip of the loop, increased both k(cat) and K(M) and gave reduced product inhibition., Increased activity was observed on amorphous cellulose, while only half, the original activity remained on crystalline cellulose. Stabilisation of, the exo-loop by the disulphide bridge enhanced the activity on both, amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3)+Glc(1)), released from cellulose, which is indicative of processive action, was, highest with Tr Cel7A wild-type enzyme and smallest with the deletion, mutant on both substrates. Based on these data it seems that the exo-loop, of Tr Cel7A has evolved to facilitate processive crystalline cellulose, degradation, which does not require significant conformational changes of, this loop.
+The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations.The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K(M) and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3)+Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.
 ==About this Structure==
-Q2E is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Hypocrea_jecorina Hypocrea jecorina] with NAG and CA as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Cellulose_1,4-beta-cellobiosidase Cellulose 1,4-beta-cellobiosidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.91 3.2.1.91] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1Q2E OCA].
+Q2E is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Hypocrea_jecorina Hypocrea jecorina] with <scene name='pdbligand=NAG:'>NAG</scene> and <scene name='pdbligand=CA:'>CA</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Cellulose_1,4-beta-cellobiosidase Cellulose 1,4-beta-cellobiosidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.91 3.2.1.91] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1Q2E OCA].
 ==Reference==
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 [[Category: Single protein]]
 [[Category: Harris, M.]]
-[[Category: Jones, T.A.]]
+[[Category: Jones, T A.]]
 [[Category: Stahlberg, J.]]
 [[Category: CA]]
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 [[Category: loop deletion]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 00:19:53 2007''
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