Purification and cellular localization of wild type and mutated dihydrolipoyltransacetylases from Azotobacter vinelandii and Escherichia coli expressed in E. coli

Abstract

Wild type dihydrolipoyltransacetylase(E2p)-components from the pyruvate dehydrogenase complex of A. vinelandii or E. coli, and mutants of A. rinelandii E2p with stepwise deletions of the lipoyl domains or the alanine-and proline-rich region between the binding and the catalytic domain have been overexpressed in E. coli TG2. The high expression of A. ~,inelandii wild type E2p (20% of cellular protein) and of a mutant enzyme with two lipoyl domains changed the properties of the inner bacterial membrane. This resulted in a solubilization of A. cinelandii E2p after degradation of the outer membrane by lysozyme without any contamination by E. coil pyruvate dehydrogenase complex (PDC) or other high-molecular-weight contaminants. The same effect could be detected for A. rinelandii E2o, an E2 which contains only one iipoyl domain, whereas almost no solubilization of A. vinelandii E2p with one lipoyl domain or of E2p consisting only of the binding and catalytic domain was found. Partial or complete deletion of the alanine-and proline-ricb sequence between the binding and the cate.lytic domain did also decrease the solubilization of the E2p-mutants after lysozyme treatment. Immunocytochemical experiments on E. coli TG2 cells expressing A. cinelandii wild type E2p indicated that the enzyme was present as a soluble protein in the cytoplasm. In contrast, overexpressed A. cinelandii E2p with deletion of all three lipoyi domains and E. coli wild type E2p aggregated intracellularly. The solubilization by lysozyme is therefore ascribed to excluded volume effects leading to changes in the properties of the inner bacterial membrane.

Reconstitution of pyruvate dehydrogenase multienzyme complexes based on chimeric core structures from Azotobacter vinelandii and Escherichia coli

“…vinelandii and E. coli E2p, the plasmids pRA282 ( A . vinelandii wild-type E2p) [3] and PAW10 (E. coli wild-type E2p) [6] were used as starting material (Fig. 1 B).…”

“…The gene encoding E2p of the PDC from E. coli was recloned into pUC9 and the resulting plasmid was named PAW10 [6]. This plasmid contains a KpnI-DraI fragment with the complete E2p gene from E. coli in the SmaI site of pUC9.…”

“…vinelandii wild-type E2p and the chimeric E2p proteins, containing the catalytic domain from A . vinelandii, were isolated from E. coli TG2 according to [6] after incubation of the E. coli TG2 cells with lysozyme. E. coli wild-type E2p and the chimeric E2p proteins, containing the catalytic domain from E. coli were purified according to [6] after disruption of the cells using a Manton-Gaulin laboratory homogenizer.…”

“…The plasmid pAW9, which was used for the overexpression and purification of E. coli wild-type E3 is described in [6].…”

“…coli TG2 cells, harbouring the appropriate plasmid, were grown at 37 "C in TY medium, containing ampicillin (75 pg/ml) and isopropyl P-D-thio-galactopyranoside (20 pg/ ml) for 20 h. For small-scale experiments ( Table 1) 10 ml of the cell culture were centrifuged for 10 min at 6000 x g . The cell pellet was dissolved in 500 pl Tris/Glc/EDTA (25 mM Tris/Cl pH 8.0, 50 mM glucose, 10 mM EDTA) and sonicated (30 W, 3 x 10 s) or incubated with lysozyme (50 pg/ml) for 60 min [6]. The supernatant after centrifugation at 11 000 x g for 10 min was used for further experiments.…”