title: Structure-function relationships and flexible tetramer assembly in pyruvate decarboxylase revealed by analysis of crystal structures.
abstract: The crystal structures of pyruvate decarboxylase from the yeast Saccharomyces uvarum and Saccharomyces cerevisiae have been determined at 2.4 and 2.3 A resolution, respectively. These structures provide details about the protein fold and domain assembly within subunits, about subunit assembly to form dimers and about dimer assembly to form tetramers. They also provide a clear picture of the active site centered on the thiamin diphosphate cofactor, and have allowed amino acids critical for catalysis and involved in stabilization of the unusual cofactor conformation to be identified. The structural information has enabled identification of the site of allosteric activation to be centered on Cys-221, and suggests that a six residue segment leading from the regulatory site to the catalytic site may be involved in transmission of a binding signal. The importance of several amino acids within this segment in the regulatory process, as well as some involved in stabilizing and activating the cofactor has been confirmed by analyzing the behavior of recombinant enzymes with single point mutations introduced at these sites. Additional structures have been determined for pyruvate decarboxylase in multiple crystal forms, some of which were obtained from crystals grown with known allosteric activators present in the media. Currently four distinct types of tetramers have been observed, with each showing a different mode of association of dimers to form the tetramers. In some of the cases involving the presence of allosteric activators drastic changes in the mode of dimer assembly to form tetramers is seen.
authors.affiliation: Biocrystallography Laboratory, VA Medical Center, PO Box 12055, University Drive C, Pittsburgh, PA 15240, USA. fureyw@vms.cis.pitt.edu
authors.forename: W
authors.initials: W
authors.lastname: Furey
authors.forename: P
authors.initials: P
authors.lastname: Arjunan
authors.forename: L
authors.initials: L
authors.lastname: Chen
authors.forename: M
authors.initials: M
authors.lastname: Sax
authors.forename: F
authors.initials: F
authors.lastname: Guo
authors.forename: F
authors.initials: F
authors.lastname: Jordan
mesh_terms: D000494:Allosteric Regulation; D001665:Binding Sites; D018360:Crystallography, X-Ray; D046911:Macromolecular Substances; D008958:Models, Molecular; D015394:Molecular Structure; D011487:Protein Conformation; D011767:Pyruvate Decarboxylase / Q000737:chemistry* / Q000378:metabolism; D012440:Saccharomyces / Q000201:enzymology; D013329:Structure-Activity Relationship; D013835:Thiamine Pyrophosphate / Q000737:chemistry* / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D011767:Pyruvate Decarboxylase; D013835:Thiamine Pyrophosphate