This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our determination and analysis of the structure of benzoylformate decarboxylase (BFD), a vitamin B1 dependent-enzyme, has begun to reveal important and unexpected lessons about this enzyme family. We have produced 9 crystal forms of BFD, which allow data collection to 1 . The structure of a covalent intermediate complex (which must be repeated to produce higher resolution data) and a inhibitor bound in the active site suggests the roles of four active-site residues (His 281, His70, Glu 28, and Ser 26). Stopped-flow spectroscopy on site-directed mutants allowed us to define roles of these residues and the order of their action. According to these His 70 and Glu 28 work together in a beginning step of the reaction, His 281 works in a later step, and, surprisingly, Ser 26 is important throughout the entire reaction. The importance of Ser 26 is supported by the crystal structures, where it is seen bound to the substrate carboxylate group that is removed in the reaction. We have identified benzoylphosphonate as a novel inactivator of BFD. Incredibly, addition of the phosphonate (containing a phosphate-carbon bond) leads to phosphorylation (a phosphate-oxygen bond) with Ser 26. Such a reaction has not been observed previously. Our results show one of the products of decarboxylation, bicarbonate, bound tightly in the active site in one of the crystal forms. Until now, bicarbonate has not been considered the product that remains in the active site. In addition, several mutants show the addition of an oxygen to the vitamin B1, demonstrating that the wild-type active site must be vigilant in preventing such reactions and allow only the correct effect to occur. In one case, what seems to be sulfate is added instead of oxygen; this should be tested with the sulfate scattering. Our future experiments will also include inspection of structures of other mutants and bound inhibitors, and Laue diffraction to understand the reaction in wild-type and mutant proteins.
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