Molecular Structure of a 9-MDa Icosahedral Pyruvate Dehydrogenase Subcomplex Containing the E2 and E3 Enzymes Using Cryoelectron Microscopy

Sriram Subramaniam,Mario J. Borgnia,Jacqueline L.S. Milne,Richard N. Perham,Bernard R. Brooks,Xiongwu Wu,Dan Shi,Jeffrey S. Lengyel

doi:10.1074/jbc.m504363200

Abstract

The pyruvate dehydrogenase multienzyme complexes are among the largest multifunctional catalytic machines in cells, catalyzing the production of acetyl CoA from pyruvate. We have previously reported the molecular architecture of an 11-MDa subcomplex comprising the 60-mer icosahedral dihydrolipoyl acetyltransferase (E2) decorated with 60 copies of the heterotetrameric (alpha(2)beta(2)) 153-kDa pyruvate decarboxylase (E1) from Bacillus stearothermophilus (Milne, J. L. S., Shi, D., Rosenthal, P. B., Sunshine, J. S., Domingo, G. J., Wu, X., Brooks, B. R., Perham, R. N., Henderson, R., and Subramaniam, S. (2002) EMBO J. 21, 5587-5598). An annular gap of approximately 90 A separates the acetyltransferase catalytic domains of the E2 from an outer shell formed of E1 tetramers. Using cryoelectron microscopy, we present here a three-dimensional reconstruction of the E2 core decorated with 60 copies of the homodimeric 100-kDa dihydrolipoyl dehydrogenase (E3). The E2E3 complex has a similar annular gap of approximately 75 A between the inner icosahedral assembly of acetyltransferase domains and the outer shell of E3 homodimers. Automated fitting of the E3 coordinates into the map suggests excellent correspondence between the density of the outer shell map and the positions of the two best fitting orientations of E3. As in the case of E1 in the E1E2 complex, the central 2-fold axis of the E3 homodimer is roughly oriented along the periphery of the shell, making the active sites of the enzyme accessible from the annular gap between the E2 core and the outer shell. The similarities in architecture of the E1E2 and E2E3 complexes indicate fundamental similarities in the mechanism of active site coupling involved in the two key stages requiring motion of the swinging lipoyl domain across the annular gap, namely the synthesis of acetyl CoA and regeneration of the dithiolane ring of the lipoyl domain.

Highlights

The pyruvate dehydrogenase multienzyme complexes are among the largest multifunctional catalytic machines in cells, catalyzing the production of acetyl CoA from pyruvate
The lipoyl domain must migrate to the active site in the acetyltransferase domain of E2 where the acetyl group is transferred to CoA, and to the active site of E3 where the pendant dihydrolipoyl group is oxidized at the expense of NADϩ to regenerate its dithiolane ring, preparing it to re-enter the active site of E1 to initiate a new cycle of catalytic events [4, 5]
A three-dimensional model of the fully occupied E2E3 subcomplex was constructed from 6054 individual molecular images computationally selected from cryoelectron micrographs, which contained well separated and randomly oriented complexes that are considerably larger and more intricate (Fig. 1, A and B) than those formed by the undecorated E2 core alone [14]

Summary

Introduction

The pyruvate dehydrogenase multienzyme complexes are among the largest multifunctional catalytic machines in cells, catalyzing the production of acetyl CoA from pyruvate. The E2E3 complex has a similar annular gap of ϳ75 Abetween the inner icosahedral assembly of acetyltransferase domains and the outer shell of E3 homodimers.

Results

Conclusion