This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The goal of the proposed research is to determine crystal structures of the transcarboxylase (TC) multienzyme complex. TC is a 1.2 million dalton biotin-dependent bacterial metabolic enzyme that has long served as a model system for human biotin-dependent metabolic enzymes; none of the human or mammalian biotin-dependent enzymes have been crystallized. Crystal structures of TC will provide insight into mechanisms of assembly and catalysis for biotin-dependent metabolic enzymes. Intact 26S TC contains 30 polypeptide chains of three types: the catalytic hexameric 12S, the catalytic dimeric 5S, and the biotinylated 1.3S which shuttles between the 12S and 5S active sites. The 26S TC can lose several 6S (5S+1.3S heterotetramers) subunits to give 18S, which is also enzymatically active. We have recently published crystal structures of the isolated 12S and 5S subunits, determined using synchrotron radiation. We propose to determine crystal structures of TC 6S, 18S, and 26S, as well as additional structures of isolated 12S and 5S, either as active site ligand complexes or mutations designed to probe enzyme mechanism or to reproduce the effect of human mutations identified in metabolic diseases.
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