On Nitroaryl Reductase Activities in SeveralClostridia

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Abstract
Crude extracts of Clostridium kluyveri, Clostridium sp. La 1, C. sporogenes and C. pasteurianum catalyze the NADH-dependent reduction of the nitro group of p-nitrobenzoate. The former 3 clostridia also use pyruvate as electron donor for this reduction. The NADH-dependent reductases were partially purified and characterized from C. kluyveri. Nitroalkyl compounds, nitrite, sulfite, sulfate and hydroxylamine are not substrates. Based on chromatographic behavior, separation pattern, yields, stability, pH optima, molecular masses and EPR studies, the 3 NADH-dependent nitroaryl group reducing enzymes in C. kluyveri (3 activities in Clostridium sp. La 1 and 2 activities in C. sporogenes) are different from alcohol dehydrogenase (EC 1.1.1.1), aldehyde dehydrogenase (EC 1.2.1.10), 3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.-) butyryl-CoA dehydrogenase (EC 1.3.99.2), 2-enoate reductase (EC 1.3.1.31), ferredoxin-NAD+ reductase (EC 1.18.1.3) and ferredoxin-NADP+ reductase (EC 1.18.1.2). The physiological roles of the nitroaryl reductases are not known. The reductase activities show losses of 80-90% during classical protein purification procedures. One of the 3 nitroaryl reductases exhibits a pH optimum of 10.5. The crude extract reveals a pH optimum at 11.5. The 1st step of the reduction reaction leads to the nitroradical anion (1 electron transfer). The electron transfer to p-nitrobenzoate is also catalyzed by ferredoxin-NAD reductase from NADH and by ferredoxin-NADP reductase from NADP. Partially purified 2-oxo-acid synthases from C. sporogenes catalyze with low rates the reduction of p-nitrobenzoate and 2-nitroethanol in the presence and absence of ferredoxin using pyruvate or 2-oxo-4-methylpentanoate as electron donors, respectively. The NADH-dependent reduction of p-nitrobenzoate accounts for .gtoreq. 70% and the 2-oxo acid-dependent reduction for .apprx. 5% of the total nitroaryl reductase activity in the clostridia. The pyridine nucleotide-dependent nitroaryl reductases are enzymes so far unknown in clostridia.