CRYSTALLOGRAPHIC STUDIES ON THE ICOSAHEDRAL CORE OF THE PYRUVATE DEHYDROGENASE MULTIENZYME COMPLEX FROM BACILLUS STEAROTHERMOPHILUS. Tina Izard^a, Richard N. Perham^b, Arie de Kok^c, Wim G. J. Hol^{a, a}Howard Hughes Medical Institute, Biomolecular Structure Center & Dept. Of Biological Structure, University of Washington, Box 357742, Seattle WA 98195-7742, USA, ^bDept. of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK, ^cDept. of Biochemistry, Agricultural University, Wageningen, The Netherlands

The bacterial and mammalian 2-oxoacid dehydrogenase multienzyme complex families catalyse the oxidative decarboxylation of 2-oxoacids (pyruvate, [[alpha]]-ketoglutarate and branched-chain 2-oxoacids) to produce the corresponding acyl-CoA and NADH. A well known member of the family is pyruvate dehydrogenase (PDH), occurring at the end of the glycolysis and providing the tricarboxylic acid cycle with acetyl-CoA. The architectural design of PDH is composed of a central core enzyme, dihydrolipoamide acetyltransferase (E2) with either octahedral (24-mer) or icosahedral (60-mer) symmetry, depending on the source of the enzyme. E2 binds the two peripheral enzymes, thiamin pyrophosphate (TPP) dependent decarboxylase (E1) and flavoenzyme lipoamide dehydrogenase (E3), leading to a molecular weight (M_r) of these systems of 5 to 10 million Da. In mammals and yeast, additional proteins are attached to the complex; the so-called protein X and a specific kinase and phosphatase. Deficiencies or malfunctioning of the complexes lead to severe pathological states such as numerous acidoses which are usually correlated with serious neurological dysfunctions.

The catalytic domain of E2 from B. stearothermophilus and Enterococcus faecalis PDH have been cloned, expressed in E. coli and purified. Of the former, crystals suitable for X-ray diffraction experiments grew within 10 days and diffract to about 4 Å resolution at cryo-temperatures. Here we describe the crystallisation of E2 from B. stearothermophilus and its preliminary analysis by X-ray crystallography.