THE CRYSTAL-STRUCTURE OF BENZOYLFORMATE DECARBOXYLASE AT 1.6 ANGSTROMRESOLUTION - DIVERSITY OF CATALYTIC RESIDUES IN THIAMIN DIPHOSPHATE-DEPENDENT ENZYMES

The crystal structure of the thiamin diphosphate (ThDP)-dependent enzy me benzoylformate decarboxylase (BFD), the third enzyme in the mandela te pathway of Pseudomonas putida, has been solved by multiple isomorph ous replacement at 1.6 Angstrom resolution and refined to an R-factor of 15.0% (free R = 18.6%). The structure of BFD has been compared to t hat of other ThDP-dependent enzymes, including pyruvate decarboxylase. The overall architecture of BFD resembles that of the other family me mbers, and cofactor- and metal-binding residues are well conserved, Su rprisingly, there is no conservation of active-site residues not direc tly bound to the cofactor. The position of functional groups in the ac tive site may be conserved, however. Three classes of metal ions have been identified in the BFD crystal structure: Ca2+ bound to the cofact or in each subunit, Mg2+ on a 2-fold axis of the tetramer, and Ca2+ at a crystal contact. The structure includes a non-proline cis-peptide b ond and an unusually long and regular polyproline type II helix that m ediates the main contact between tetramers in the crystal. The high-qu ality electron-density map allowed the correction of errors totaling m ore than 10% of the amino acid sequence, which had been predicted from the reported sequence of the mdlC gene. Analysis of the BFD structure suggests that requirements for activation of the cofactor, the nature of the reaction intermediates, and architectural considerations relat ing to the protein fold have been dominant forces in the evolution of ThDP-dependent enzymes.