Redesigning the active site of transaldolase TalB from Escherichia coli: new variants with improved affinity towards nonphosphorylated substrates.
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.
Schneider
Sarah
S
Institute of Microbiology, Universität Stuttgart, Allmandring 31, Germany.
Gutiérrez
Mariana
M
Sandalova
Tatyana
T
Schneider
Gunter
G
Clapés
Pere
P
Sprenger
Georg A
GA
Samland
Anne K
AK
D019943:Amino Acid Substitution; D001665:Binding Sites; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004098:Dihydroxyacetone / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D005632:Fructose / Q000096:biosynthesis; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D013379:Substrate Specificity; D014153:Transaldolase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D005632:Fructose; D014153:Transaldolase; D004098:Dihydroxyacetone