3cwn

Publication Abstract from PubMed

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.

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.,Schneider S, Sandalova T, Schneider G, Sprenger GA, Samland AK J Biol Chem. 2008 Oct 31;283(44):30064-72. Epub 2008 Aug 7. PMID:18687684^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.