Replacement of a phenylalanine by a tyrosine in the active site confers fructose-6-phosphate aldolase activity to the transaldolase of Escherichia coli and human origin.
Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe(178) to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody.
Schneider
Sarah
S
Institute of Microbiology, Universität Stuttgart, 70550 Stuttgart, Germany.
Sandalova
Tatyana
T
Schneider
Gunter
G
Sprenger
Georg A
GA
Samland
Anne K
AK
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002474:Cell-Free System; D004926:Escherichia coli / Q000201:enzymology*; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry*; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014153:Transaldolase / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
D014443:Tyrosine; D010649:Phenylalanine; D014153:Transaldolase; D005634:Fructose-Bisphosphate Aldolase