The catalytic domain of dihydrolipoyl acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

Abstract

A sub-gene encoding the catalytic (acetyltransferase) domain (E2pCD) comprising residues 173-427 of the dihydrolipoyl acetyltransferase (E2p) chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was expressed in Escherichia coli. The product assembled to form the characteristic icosahedral (60-mer) core structure with full catalytic activity. The K m values for dihydrolipoamide and acetyl-CoA were 1.2 mM and 13 jiM, respectively. Dissociation of the icosahedral E2pCD into monomers by exposure to guanidine hydrochloride and the subsequent reassociation by gradual removal of the denaturing agent demonstrated the ability of the polypeptide chain to fold and reassemble in the absence of chaperonins.

Optimal Determination of Particle Orientation, Absolute Hand, and Contrast Loss in Single-particle Electron Cryomicroscopy

“…Purified B. stearothermophilus dihydrolipoyl acetyltransferase inner core, a 60-mer of the 28 kDa acetyltransferase domain comprising residues 173-427 of the E2 chain, 35 was a gift from Gonzalo J. Domingo and Richard Perham. Samples (3 mg/ml) in 50 mM sodium phosphate buffer, pH 7.0 were stored at 270 8C, and were diluted 1:10 (v/v) in PBS (phosphate-buffered saline) immediately prior to use.…”

Principles of quasi-equivalence and Euclidean geometry govern the assembly of cubic and dodecahedral cores of pyruvate dehydrogenase complexes

“…Crystals of the catalytic domain E2p of B. stearothermophilus (residues 173-427; ref. 17) were obtained using vapor diffusion by equilibrating 6 l of a 10 mg/ml of purified protein solution in a 50 mM Tris⅐HCl buffer at pH 8.0 against 6 l of a reservoir solution containing 20% 2-methyl-2,4-pentanediol (MPD), 100 mM Tris⅐HCl (pH 7.0), and 5% polyethylene glycol (M r ϭ 1,000). Crystals were cryoprotected by increasing the MPD concentration to 35% while keeping the buffer and additive unchanged.…”

Thermostability and molecular encapsulation within an engineered caged protein scaffold

“…Our proteins were produced using a previously reported protocol for E2 purification as a departure point (Allen and Perham, 1997). E. coli strain BL21(DE3), transformed with the wild-type or mutant gene, was cultured in Luria-Bertani medium supplemented with ampicillin (100 mg/mL) under constant shaking at 378C.…”

“…Therefore, we have investigated both the feasibility of introducing non-native, functional modifications, as well as the impact of these alterations on the structural and thermal stabilities of our engineered scaffold. While the catalytic mechanisms and biology of the pyruvate dehydrogenase complex from Bacillus stearothermophilus has been well-studied (Allen and Perham, 1997;Domingo et al, 2001Domingo et al, , 2003Izard et al, 1999;Jung et al, 2002;Lessard et al, 1998;Milne et al, 2002Milne et al, , 2006Perham, 1991), the potential application of its E2 structural scaffold as a molecular carrier has not been investigated.…”