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1a47

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{{STRUCTURE_1a47| PDB=1a47 | SCENE= }}
{{STRUCTURE_1a47| PDB=1a47 | SCENE= }}
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'''CGTASE FROM THERMOANAEROBACTERIUM THERMOSULFURIGENES EM1 IN COMPLEX WITH A MALTOHEXAOSE INHIBITOR'''
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===CGTASE FROM THERMOANAEROBACTERIUM THERMOSULFURIGENES EM1 IN COMPLEX WITH A MALTOHEXAOSE INHIBITOR===
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==Overview==
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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.
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{{ABSTRACT_PUBMED_9488711}}
==About this Structure==
==About this Structure==
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[[Category: Substrate]]
[[Category: Substrate]]
[[Category: Thermostable]]
[[Category: Thermostable]]
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 09:47:14 2008''
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jun 30 16:02:17 2008''

Revision as of 13:02, 30 June 2008

Image:1a47.png

Template:STRUCTURE 1a47

CGTASE FROM THERMOANAEROBACTERIUM THERMOSULFURIGENES EM1 IN COMPLEX WITH A MALTOHEXAOSE INHIBITOR

Template:ABSTRACT PUBMED 9488711

About this Structure

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

Reference

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

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