Crystallographic studies of transaldolase : implications for the enzymatic mechanism and the evolution of class I aldolases

Sammanfattning: Crystallographic studies of transaldolase Implications for the enzymatic mechanism and the evolution of class I aldolases Jia Jia Transaldolase catalyzes the reversible transfer of a dihydroxyacetone moiety froma ketose donor to an aldose acceptor by forming a Schiff-base intermediate betweena Iysine residue and dihydroxyacetone. It belongs to the class I aldolase farnily.A common mechanistic feature of the members of this enzyme family is the formationof a covalent intermediate between an active site Iysine residue and the substrateduring catalysis. The three-dimensional structure of recombinant transaldolase B from E.coli hasbeen 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 of20.1% 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. Thefold of transaldolase is similar to other enzyme structures in the class I aldolasefamily. Comparison of these structures suggests that a circular permutation has occurredin the ancestral aldolase gene. This observation provides the first structural evidencefor a naturally occuring circular permutation in an a/B barrel protein. The structure of a trapped Schiff-base intermediate complex of this enzyme hasbeen determined at 2.2 A resolution and refined to R-factor of 20.4% and R-free of24.4%. Tbe structure of the complex provides direct crystallographic evidence forthe 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 mechanismare: Lys 132 acts as the Schiff base forming residue, and two acidic groups, Glu96and Aspl7 and a catalytic water molecule are involved in proton transfer during thereaction. Several functionally important residues at the active site and dimer interfacehave been mutated by site-directed mutagenesis and the mutants have been analyzedby crystallography. The structural analysis confirmed the mutations and showed thatno unintended structural changes were introduced. The kinetic properties of thesemutant enzymes are consistent with the proposed roles for these residues in catalysis. Keywords: transaldolase, aldolase, Schiff-base intermediate, protein crystallography,site directed mutagenesis, catalytic mechanism, circular permutation. Jia Jia, Molecular Structural Biology, Department of Medical Biochemistry andBiophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden ISBN 91-628-2718-9 Printed in Sweden by Akademitryck AB, Edsbruk