Leucine dissimilation to isovaleric and isocaproic acids by cell suspensions of amino acid fermenting anaerobes: the Stickland reaction revisited

Freshly prepared cell suspensions of Clostridia (Clostridium bifermentans, C. botulinum proteolytic type A, C. difficile, C. sordellii, and C. sporogenes) and Peptostreptococcus anaerobius converted leucine to isovaleric (iV) and isocaproic (iC) acids in the absence of other amino acids. The optimal pH for conversion was between 8 and 9 at 37 °C. The stoichiometry of reaction was compatible with that expected for the Stickland reaction, as the ratio of iV to iC was 1:2, the amount of CO2 produced was equivalent to that of iV, and ammonium ion concentrations were equal to the total C5 and C6 acids formed. The presence of alanine and valine (proton donors in the Stickland reaction) in incubations effectively increased the concentration of iC at the expense of iV production, implying that leucine acted here primarily as a proton acceptor. Glycine and proline (proton acceptors) stimulated both iV and iC production from leucine, but increases in iV concentrations were proportionately greater than for iC, so that leucine was primarily a proton donor in the presence of proton acceptors. Glucose stimulated the conversion of leucine to volatile fatty acids but favoured iC production. Production of iC from leucine was inhibited by surface active compounds (cetyltrimethylammonium bromide and desoxycholate) as well as arsenite and iodoacetate. The redox dyes methyl viologen and phenosafranine inhibited iC production more severely than iV production, as did the nitroimidazole antimicrobial agent, metronidazole.