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Redesigning the Active Site of Transaldolase TalB from Escherichia coli : New Variants with Improved Affinity towards Nonphosphorylated Substrates
Author(s) -
Schneider Sarah,
Gutiérrez Mariana,
Sandalova Tatyana,
Schneider Gunter,
Clapés Pere,
Sprenger Georg A.,
Samland Anne K.
Publication year - 2010
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200900720
Subject(s) - transaldolase , aldolase a , chemistry , active site , saturated mutagenesis , aldol reaction , glycolaldehyde , stereochemistry , escherichia coli , protein engineering , biochemistry , biocatalysis , directed evolution , enzyme , mutant , pentose phosphate pathway , reaction mechanism , glycolysis , gene , catalysis
Abstract Recently, we reported on a transaldolase B variant (TalB F178Y) that is able to use dihydroxyacetone (DHA) as donor in aldol reactions. In a second round of protein engineering, we aimed at improving the affinity of this variant towards nonphosphorylated acceptor aldehydes, that is, glyceraldehyde (GA). The anion binding site was identified in the X‐ray structure of TalB F178Y where a sulfate ion from the buffer was bound in the active site. Therefore, we performed site‐directed saturation mutagenesis at three residues forming the putative phosphate binding site, Arg181, Ser226 and Arg228. The focused libraries were screened for the formation of D ‐fructose from DHA and d,l ‐GA by using an adjusted colour assay. The best results with respect to the synthesis of D ‐fructose were achieved with the TalB F178Y/R181E variant, which exhibited an at least fivefold increase in affinity towards d,l ‐GA ( K M =24 m M ). We demonstrated that this double mutant can use D ‐GA, glycolaldehyde and the L ‐isomer, L ‐GA, as acceptor substrates. This resulted in preparative synthesis of D ‐fructose, D ‐xylulose and L ‐sorbose when DHA was used as donor. Hence, we engineered a DHA‐dependent aldolase that can synthesise the formation of polyhydroxylated compounds from simple and cheap substrates at preparative scale.