3bmv: Difference between revisions

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{{STRUCTURE_3bmv|  PDB=3bmv  |  SCENE=  }}  
{{STRUCTURE_3bmv|  PDB=3bmv  |  SCENE=  }}  


'''Cyclodextrin glycosyl transferase from Thermoanerobacterium thermosulfurigenes EM1 mutant S77P'''
===Cyclodextrin glycosyl transferase from Thermoanerobacterium thermosulfurigenes EM1 mutant S77P===




==Overview==
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Thermoanaerobacterium thermosulfurigenes cyclodextrin glucanotransferase primarily catalyzes the formation of cyclic alpha-(1,4)-linked oligosaccharides (cyclodextrins) from starch. This enzyme also possesses unusually high hydrolytic activity as a side reaction, thought to be due to partial retention of ancestral enzyme function. This side reaction is undesirable since it produces short saccharides that are responsible for the breakdown of the cyclodextrins formed, thus limiting the yield of cyclodextrins produced. To reduce the competing hydrolysis reaction, while maintaining the cyclization activity, we applied directed evolution, introducing random mutations throughout the cgt gene by error-prone PCR. Mutations in two residues, Ser-77 and Trp-239, on the outer region of the active site, lowered the hydrolytic activity up to 15-fold with retention of cyclization activity. In contrast, mutations within the active site could not lower hydrolytic rates, indicating an evolutionary optimized role for cyclodextrin formation by residues within this region. The crystal structure of the most effective mutant, S77P, showed no alterations to the peptide backbone. However, subtle conformational changes to the side chains of active site residues had occurred, which may explain the increased cyclization/hydrolysis ratio. This indicates that secondary effects of mutations located on the outer regions of the catalytic site are required to lower the rates of competing side reactions, whilst maintaining primary catalytic function. Subsequent functional analysis of various glucanotransferases from the superfamily of glycoside hydrolases also suggests a gradual evolutionary progression of these enzymes from a common 'intermediate-like' ancestor toward specific transglycosylation activity.
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{{ABSTRACT_PUBMED_18422488}}


==About this Structure==
==About this Structure==
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==Reference==
==Reference==
Elimination of competing hydrolysis and coupling side reactions of a cyclodextrin glucanotransferase by directed evolution., Kelly RM, Leemhuis H, Rozeboom HJ, van Oosterwijk N, Dijkstra BW, Dijkhuizen L, Biochem J. 2008 Apr 21;. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/18422488 18422488]
Elimination of competing hydrolysis and coupling side reactions of a cyclodextrin glucanotransferase by directed evolution., Kelly RM, Leemhuis H, Rozeboom HJ, van Oosterwijk N, Dijkstra BW, Dijkhuizen L, Biochem J. 2008 Apr 21;. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/18422488 18422488]
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:[http://www.ncbi.nlm.nih.gov/pubmed/9488711 9488711]
Crystal structure at 2.3 A resolution and revised nucleotide sequence of the thermostable cyclodextrin glycosyltransferase from Thermonanaerobacterium thermosulfurigenes EM1., Knegtel RM, Wind RD, Rozeboom HJ, Kalk KH, Buitelaar RM, Dijkhuizen L, Dijkstra BW, J Mol Biol. 1996 Mar 1;256(3):611-22. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/8604143 8604143]
[[Category: Cyclomaltodextrin glucanotransferase]]
[[Category: Cyclomaltodextrin glucanotransferase]]
[[Category: Single protein]]
[[Category: Single protein]]
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[[Category: Thermostable]]
[[Category: Thermostable]]
[[Category: Transferase]]
[[Category: Transferase]]
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Wed May 28 09:19:47 2008''
 
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun Jul 27 13:19:08 2008''

Revision as of 10:19, 27 July 2008

File:3bmv.png

Template:STRUCTURE 3bmv

Cyclodextrin glycosyl transferase from Thermoanerobacterium thermosulfurigenes EM1 mutant S77P

Template:ABSTRACT PUBMED 18422488

About this Structure

3BMV is a Single protein structure of sequence from Thermoanaerobacterium thermosulfurigenes. Full crystallographic information is available from OCA.

Reference

Elimination of competing hydrolysis and coupling side reactions of a cyclodextrin glucanotransferase by directed evolution., Kelly RM, Leemhuis H, Rozeboom HJ, van Oosterwijk N, Dijkstra BW, Dijkhuizen L, Biochem J. 2008 Apr 21;. PMID:18422488

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

Crystal structure at 2.3 A resolution and revised nucleotide sequence of the thermostable cyclodextrin glycosyltransferase from Thermonanaerobacterium thermosulfurigenes EM1., Knegtel RM, Wind RD, Rozeboom HJ, Kalk KH, Buitelaar RM, Dijkhuizen L, Dijkstra BW, J Mol Biol. 1996 Mar 1;256(3):611-22. PMID:8604143

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