Wild-type and mutant forms of the pyruvate dehydrogenase multienzyme complex from Bacillus subtilis

Abstract

A simple procedure is described for the purification of the pyruvate dehydrogenase complex and dihydrolipoamide dehydrogenase from Bacillus subtilis. The method is rapid and applicable to small quantities of bacterial cells. The purified pyruvate dehydrogenase complex (s0(20),w = 73S) comprises multiple copies of four different types of polypeptide chain, with apparent Mr values of 59 500, 55 000, 42 500 and 36 000: these were identified as the polypeptide chains of the lipoate acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3) and the two types of subunit of the pyruvate decarboxylase (E1) components respectively. Pyruvate dehydrogenase complexes were also purified from two ace (acetate-requiring) mutants of B. subtilis. That from mutant 61142 was found to be inactive, owing to an inactive E1 component, which was bound less tightly than wild-type E1 and was gradually lost from the E2E3 subcomplex during purification. Subunit-exchange experiments demonstrated that the E2E3 subcomplex retained full enzymic activity, suggesting that the lesion was limited to the E1 component. Mutant 61141R elaborated a functional pyruvate dehydrogenase complex, but this also contained a defective E1 component, the Km for pyruvate being raised from 0.4 mM to 4.3 mM. The E1 component rapidly dissociated from the E2E3 subcomplex at low temperature (0-4 degrees C), leaving an E2E3 subcomplex which by subunit-exchange experiments was judged to retain full enzymic activity. These ace mutants provide interesting opportunities to analyse defects in the self-assembly and catalytic activity of the pyruvate dehydrogenase complex.

Secretory S complex of Bacillus subtilis: sequence analysis and identity to pyruvate dehydrogenase

“…The B. subtilis and B. stearothermophilus PDH is a complex consisting of four protein subunits. The molecular weights of the B. subtilis PDH subunits are 42,000, 38,000, 66,000, and 63,000 on a 12.5% acrylamide gel with Trisglycine buffer and 42,000, 36,000, 59,000, and 54,000 on a 7.5% gel with phosphate buffer (32). Thus, the sizes are reasonably similar to those of the S-complex proteins.…”

“…Some properties of the S complex are similar to those of the PDH, which has been purified from B. subtilis (32,69) and from a closely related species, B. stearothermophilus (29,30). The B. subtilis and B. stearothermophilus PDH is a complex consisting of four protein subunits.…”

“…Based on the homology data, we conclude that the S complex may be identified as the B. subtilis PDH. The B. subtilis PDH has been previously isolated and shown to contain four subunits with very similar sizes to those of the S complex (32). The B. subtilis PDH complex also possesses BCDH activity (41).…”

“…The B. subtilis PDH exists as a complex which has been reported to sediment at 73S (32). The B. stearothermophilus PDH complex sediments at 75S, and in electron microscopy these complexes have a diameter of 40 nm (29).…”

“…However, there are B. subtilis strains with mutations in the El and E3 subunit genes. In an aceAl mutant, both Ela and El subunits are present but they have low binding affinity to the E2-E3 subcomplex (32). A citL22 mutant has 6% of the E3 activity left but does not possess measurable PDH and OGDH activities (31).…”