The crystal structure of the thiamin diphosphate (ThDP)-dependent enzyme benzoylformate decarboxylase (BFD), the third enzyme in the mandelate pathway of Pseudomonas putida, has been solved by multiple isomorphous replacement at 1.6 A resolution and refined to an R-factor of 15.0% (free R = 18.6%). The structure of BFD has been compared to that of other ThDP-dependent enzymes, including pyruvate decarboxylase. The overall architecture of BFD resembles that of the other family members, and cofactor- and metal-binding residues are well conserved. Surprisingly, there is no conservation of active-site residues not directly bound to the cofactor. The position of functional groups in the active site may be conserved, however. Three classes of metal ions have been identified in the BFD crystal structure: Ca2+ bound to the cofactor 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 bond and an unusually long and regular polyproline type II helix that mediates the main contact between tetramers in the crystal. The high-quality electron-density map allowed the correction of errors totaling more 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 relating to the protein fold have been dominant forces in the evolution of ThDP-dependent enzymes.

No abstract

The acetylenic analogue of adenosine 9-(5',6'-dideoxy-beta-D-ribo-hex-5'-ynofuranosyl)adenine has been synthesized, and its behavior as an inhibitor of bovine S-adenosylhomocysteine hydrolase has been examined. Incubation of the enzyme with excess inhibitor caused a time-dependent, irreversible inactivation of the enzyme that was accompanied by the reduction of two equivalents of NAD+ to NADH and the loss of the two remaining equivalents of NAD+. With use of radiolabeled inhibitor, it was established that 4 equiv of the acetylenic analog bind irreversibly to the enzyme and that 4 equiv were required to inactivate the enzyme completely. The inactivated enzyme could not be reactivated by incubation with NAD+. Denaturation studies revealed that 2 equiv of the inhibitor are bound more tightly to the enzyme than the remainder, suggesting the formation of a covalent linkage between the oxidized inhibitor and the enzyme. The putative covalent linkage was found to be acid sensitive but stable to mild base. The linkage could not be stabilized by treatment of the enzyme-inhibitor complex with either borohydride or cyanoborohydride. A Kl of 173 nM was measured for the inhibitor, making it one of the more potent inhibitors that have been reported. The enzyme used in these studies was isolated by modification of an affinity chromatography method reported by Narayanan and Borchardt [(1988) Biochim. Biophys. Acta 965, 22-28]. The affinity chromatography unexpectedly led to the isolation of two forms of the enzyme. The major form contained 4.0 mol of nucleotide cofactor/mol of enzyme tetramer, while the minor form carried only 2.0 mol/tetramer.

An interesting new approach to capillary affinity gel electrophoresis (CAGE) has been developed for the selective capture and separation of homopolymer and heteropolymer oligonucleotides. The combination of selectivity of bioaffinity recognition and high-resolution power of capillary gel electrophoresis allows the on-line sequence- and size-specific separation of oligonucleotides. Both rigid gel formulations and viscous replaceable polymer solutions having user-defined, single-stranded oligonucleotides covalently attached as recognition sequences are used. Contrary to most known affinity systems in capillary electrophoresis, which operate in a continuous mode, binding and release are accomplished in two steps, effectively separating the affinity from the separation step. At low temperature, oligonucleotides with complementary sequences in the analyte solution will bind to the immobilized recognition sequence while unrelated oligonucleotides will continue to migrate. This step is a preseparation, removing all nonspecific solutes from the sample. The release of the bound solutes is achieved at elevated temperature, allowing a probe of cross-reactivity for a given biorecognition element. Applications for high-resolution separations of short oligonucleotides and their mismatches are shown, and the potential for on-line preconcentration and separation of dilute analyte solutions, thus effectively enhancing the sensitivity, is demonstrated.