3KOF

Crystal structure of the double mutant F178Y/R181E of E.coli transaldolase B

Summary for 3KOF

• Entry DOI: 10.2210/pdb3kof/pdb
• Related: 1I2N 1I2O 1I2P 1I2Q 1I2R 1ONR 1UCW 3CWN
• Descriptor: Transaldolase B, SULFATE ION (3 entities in total)
• Functional Keywords: transaldolase, aldolase, directed evolution, cytoplasm, pentose shunt, transferase
• Biological source: Escherichia coli K-12
• Cellular location: Cytoplasm (Probable): P0A870
• Total number of polymer chains: 2
• Total formula weight: 75281.17

Authors

Schneider, S.,Gutierrez, M.,Sandalova, T.,Schneider, G.,Clapes, P.,Sprenger, G.A.,Samland, A.K. (deposition date: 2009-11-13, release date: 2010-02-23, Last modification date: 2023-09-06)

Primary citation

Schneider, S.,Gutierrez, M.,Sandalova, T.,Schneider, G.,Clapes, P.,Sprenger, G.A.,Samland, A.K.
Redesigning the Active Site of Transaldolase TalB from Escherichia coli: New Variants with Improved Affinity towards Nonphosphorylated Substrates.
Chembiochem, 11:681-690, 2010

PubMed 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 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 polyhydroxylated compounds from simple and cheap substrates at preparative scale.

PubMed: 20148428
DOI: 10.1002/cbic.200900720

Experimental method

X-RAY DIFFRACTION (1.9 Å)