Journal
CHEMBIOCHEM
Volume 11, Issue 5, Pages 681-690Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.200900720
Keywords
biocatalysis; protein engineering; saturation mutagenesis; substrate specificity; transaldolase
Funding
- Deutsche Forschungsgemeinschaft [SPP1170/Sp503/4-2]
- Swedish Research Council
- Spanish MCINN [CTQ2006-01345/BQU]
- Generalitat de Catalunya [DURSI 2005-SGR-00698]
- ESF [CM0701]
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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 mm). 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 poly-hydroxylated compounds from simple and cheap substrates at preparative scale.
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