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Protocatechuate 3,4-dioxygenase (EC 1.13.1.3) was purified to homogeneity from extracts of Azotobacter vinelandii. The molecular weight of the oligomeric protein was estimated to be 510 000 by gel filtration and 480 000 by ultracentrifugation. The oligomer appears to be formed by association of equal amounts of nonidentical subunits which were estimated by sodium dodecyl sulfate gel electrophoresis to have respective molecular weights of 23 300 and 25 250. Ten gram-atoms of iron was associated with each mol of oligomer. Therefore, the enzyme appears to be a decamer with the structure 10(alpha beta Fe). T-HE AMINO ACID COMPOSITION OF Azotobacter protocatechuate oxygenase closely resembles the amino acid compositions of protocatechuate 3,4-dioxygenases from Pseudomonas aeruginosa and Thiobacillus sp. These proteins from P. aeruginosa and P. putida are known to be formed by association of nonidentical subunits of a physical size similar to the subunits of the Azotobacter enzyme. Furthermore, antisera prepared against the Azotobacter oxygenase cross-reacted strongly with the isofunctional enzymes from the two fluorescent Pseudomonas species. A weak immunological cross-reaction was observed when the antisera were tested against protocatechuate 3,4-dioxygenase from Acinetobacter calcoaceticus. The results favor the conclusion that the bacterial protocatechuate 3,4-dioxygenases were derived from a common ancestral protein.

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Fractionated extracts of Propionibacterium shermanii were found to contain the readily dissociable type of fatty acid synthase complex. For optimal activity the partially purified synthase required the presence of acetyl-CoA, malonyl-CoA, NADH and NADPH as well as a cell-free fraction containing the acyl carrier protein (ACP). When ACP-containing fractions obtained from P. shermanii grown in the presence of [ l-'4C]pantothenate were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the radioactivity was located in a peak corresponding to a molecular weight of approx. 8500-9000. The biological activity of the ACP was relatively heat-stable and trypsin-resistant. When the labelled ACP-containing fraction was subjected to gel filtration on a calibrated Sephadex G-75 column, a radioactive peak corresponding to a molecular weight of 22 000 was obtained that could participate in the 14C0,-malonyl-CoA exchange reaction in the presence of the other requisite components.

An acyl coenzyme A (CoA) carboxylase, which catalyzes the adenosine triphosphate-dependent fixation of C02 into acetyl-, propionyl-, and butyryl-CoA, was detected in fractionated cell extracts of Propionibacterium shermanii. Catalytic activity was inhibited by avidin but was unaffected by avidin pretreated with excess biotin. The carboxylase levels detected were relatively small and were related to cellular growth. Maximal carboxylase activity was detected in cells grown for about 96 h. Thereafter, the activity declined rapidly. Optimal C02 fixation occurred at pH 7.5. Other parameters of the assay system were optimized, and the apparent Km values for substrates were determined. The end product of the reaction (with acetyl-CoA as the substrate) was identified as malonyl-CoA. The stoichiometry of the reaction was such that, for every mole of acetyl-CoA and adenosine triphosphate consumed, 1 mol each of malonyl-CoA, adenosine diphosphate, and orthophosphate was formed. These data provide the first evidence for the presence of another biotin-containing enzyme, an acyl-CoA carboxylase, in these bacteria in addition to the well-characterized methylmalonyl-CoA carboxyltransferase. 4.1.1.38). The combined actions ofthese enzymes can (10, 25) result in the formation of malonyl-CoA via the following sequence of reactions: 933 on August 1, 2020 by guest