1a47
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CGTASE FROM THERMOANAEROBACTERIUM THERMOSULFURIGENES EM1 IN COMPLEX WITH A MALTOHEXAOSE INHIBITOR
OverviewOverview
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.
About this StructureAbout this Structure
1A47 is a Single protein structure of sequence from Thermoanaerobacterium thermosulfurigenes with CA as ligand. Active as Cyclomaltodextrin glucanotransferase, with EC number 2.4.1.19 Structure known Active Sites: AM1, AM2, AM3, AP1, AP2, AP3, BS3, CA1 and CA2. Full crystallographic information is available from OCA.
ReferenceReference
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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