Abstract
Transaldolase catalyzes the reversible transfer of a dihydroxyacetone moiety from a ketose donor to an aldose acceptor by forming a Schiff-base intermediate between a Iysine residue and dihydroxyacetone. It belongs to the class I aldolase family. A common mechanistic feature of the members of this enzyme family is the formation of a covalent intermediate between an active site Iysine residue and the substrate during catalysis.
The three-dimensional structure of recombinant transaldolase B from E.coli has been determined at 1.87 A resolution using the multiple isomorphous replacement method. The current model comprises residues 2-317 and has been refined to an R-factor of 20.1% and R free of 23.4%. The overall structure consists of a single domain, ana/B barrel. The active site is located at the C-terminal end of the B strands. The fold of transaldolase is similar to other enzyme structures in the class I aldolase family. Comparison of these structures suggests that a circular permutation has occurred in the ancestral aldolase gene. This observation provides the first structural evidence for a naturally occurring circular permutation in an a/B barrel protein.
The structure of a trapped Schiff-base intermediate complex of this enzyme has been determined at 2.2 A resolution and refined to R-factor of 20.4% and R-free of 24.4%. Tbe structure of the complex provides direct crystallographic evidence for the formation of a Schiff base intermediate in this enzyme family. Based on the structure, a reaction mechanism for transaldolase is proposed. The main features of this mechanism are: Lys 132 acts as the Schiff base forming residue, and two acidic groups, Glu96 and Aspl7 and a catalytic water molecule are involved in proton transfer during the reaction.
Several functionally important residues at the active site and dimer interface have been mutated by site-directed mutagenesis and the mutants have been analyzed by crystallography. The structural analysis confirmed the mutations and showed that no unintended structural changes were introduced. The kinetic properties of these mutant enzymes are consistent with the proposed roles for these residues in catalysis.
