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1pez

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{{STRUCTURE_1pez| PDB=1pez | SCENE= }}
{{STRUCTURE_1pez| PDB=1pez | SCENE= }}
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'''Bacillus circulans strain 251 mutant A230V'''
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===Bacillus circulans strain 251 mutant A230V===
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==Overview==
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Cyclodextrin glycosyltransferase (CGTase) preferably catalyzes transglycosylation reactions, whereas many other alpha-amylase family enzymes are hydrolases. Despite the availability of three-dimensional structures of several transglycosylases and hydrolases of this family, the factors that determine the hydrolysis and transglycosylation specificity are far from understood. To identify the amino acid residues that are critical for the transglycosylation reaction specificity, we carried out error-prone PCR mutagenesis and screened for Bacillus circulans strain 251 CGTase mutants with increased hydrolytic activity. After three rounds of mutagenesis the hydrolytic activity had increased 90-fold, reaching the highest hydrolytic activity ever reported for a CGTase. The single mutation with the largest effect (A230V) occurred in a residue not studied before. The structure of this A230V mutant suggests that the larger valine side chain hinders substrate binding at acceptor subsite +1, although not to the extent that catalysis is impossible. The much higher hydrolytic than transglycosylation activity of this mutant indicates that the use of sugar acceptors is hindered especially. This observation is in favor of a proposed induced-fit mechanism, in which sugar acceptor binding at acceptor subsite +1 activates the enzyme in transglycosylation [Uitdehaag et al. (2000) Biochemistry 39, 7772-7780]. As the A230V mutation introduces steric hindrance at subsite +1, this mutation is expected to negatively affect the use of sugar acceptors. Thus, the characteristics of mutant A230V strongly support the existence of the proposed induced-fit mechanism in which sugar acceptor binding activates CGTase in a transglycosylation reaction.
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{{ABSTRACT_PUBMED_12809508}}
==About this Structure==
==About this Structure==
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[[Category: Glycosyltransferase]]
[[Category: Glycosyltransferase]]
[[Category: Transferase]]
[[Category: Transferase]]
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 05:00:40 2008''
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 29 12:11:54 2008''

Revision as of 09:11, 29 July 2008

Image:1pez.png

Template:STRUCTURE 1pez

Bacillus circulans strain 251 mutant A230V

Template:ABSTRACT PUBMED 12809508

About this Structure

1PEZ is a Single protein structure of sequence from Bacillus circulans. Full crystallographic information is available from OCA.

Reference

Conversion of cyclodextrin glycosyltransferase into a starch hydrolase by directed evolution: the role of alanine 230 in acceptor subsite +1., Leemhuis H, Rozeboom HJ, Wilbrink M, Euverink GJ, Dijkstra BW, Dijkhuizen L, Biochemistry. 2003 Jun 24;42(24):7518-26. PMID:12809508

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