Isocitrate Lyase and Adenosine Triphosphate Malate Lyase as Key Enzymes for the Methylotrophic Growth of Bacterium 5H2

Abstract

During an investigation of the carbon-assimilation pathway of C1 compounds by the facultative methylotroph bacterium 5H2 (Hampton & Zatman, 1973) isocitrate lyase (EC 4.1.3.1) activities in cell-free extracts of organisms grown on a variety of substrates were measured by the method of Dixon & Kornberg (1959). The method of Kramer et a/. (1959) was used to confirm that a product of the reaction was glyoxylate, the phenylhydrazone of the latter producing the intensely red-coloured 1,Sdiphenylformazancarboxylic acid on treatment with ferricyanide. The isocitrate lyase specific activities of bacterium 5H2 grown on tetramethylammonium chloride, trimethylamine, dimethylamine, methylamine, and on acetate, were respectively, 72, 84, 103, 95 and 150munits/mg of protein, whilst the values for extracts of cells grown on fructose, glutamate and on glycerol were respectively 1.9, I .5 and 2.0munits/mg of protein. These results suggest that isocitrate lyase is important for the inethylotrophic growth of bacterium 5H2 and, not unexpectedly, for growth on acetate. High specific activities of malate synthase (EC 4.1.3.2), measured by method of Dixon & Kornberg (1959), were found in cell-free extracts of bacterium 5H2 grown on CI as well as non-C1 substrates. The results of the following induction experiment confirmed the importance of isocitrate lyase for the growth of bacterium 5H2 on trimethylamine. Glycerol-grown cells, shaken at 30°C in a mineral medium to which 0.2% trimethylarnine had been added, showed no significant growth for the first 6h but the specific activity of isocitrate lyase increased from 2.0 to approximately 45 munits/mg of protein during this period. The specific activity of isocitrate lyase reached a maximum value of approximately lOOmunits/mg of protein about 3 h later, by which time the bacteria were growing exponentially. In a simultaneous experiment in which glycerol-grown cells were shaken at 30°C in the mineral medium containing 0.2% glycerol, exponential growth occurred immediately and there was no increase in the specific activity of the isocitrate lyase throughout the experiment. Present evidence (Salem et al., 1972; Dunstan el a[., 1972; Quayle, 1972) suggests that, in organisms which use the serine pathway of C1 assimilation, regeneration of the glycine acceptor for C1 units arises via the cleavage of a C4 metabolite. During a search for such a C, cleavage reaction, it was observed that crude cell-free extracts of trimethylamine-grown bacterium 5H2 (French press, 35000g supernatant) catalyse, in the presence of hydroxylamine, an ATP-, CoAand Mgz+-dependent formation of acetylhydroxamate from L-malate. Further investigation indicated that the reaction is mediated by a malate cleavage enzyme (ATP malate lyase) which splits L-malate to acetylCoA and glyoxylate and which is similar to the malate lyase cleavage activity described by Tuboi & Kikuchi (1963) in Rhodopseirclomorias spheroicks grown phototrophically with malate. Our reaction mixture ( I ml total volume) contained (pniol): Tris-HC1 buffer pH7.8, 200; ATP, 10; CoA, 0.1; MgCI2, 5 ; L-malate, 100; NH20H,HC1 freshly neutralized, 500; crude cell-free extract (I-2mg of protein). This was incubated without shaking at 30°C, usually for 30min, the reaction being stopped by heating at 100°C for 2min, and a sample of the supernatant being chromatographed on paper by the method of Tuboi & Kikuchi (1962) to identify the hydroxarnates. Acetyl-, succinyland malyl-hydroxamic acids for marker spots on the chromatograms were prepared from their respective anhydrides by the method of Lipmann & Tuttle (1945). A measure of the total hydroxamic acids in the reaction mixture was obtained by the FeCI, method of Lipmann & Tuttle (1945). The major FeCI,-positive spot on the chromatogranis was identified as acetylhydroxamic acid, and a minor spot accounting for approximately 5 % of the total hydroxamate, was identified as malylhydroxamic acid. Glyoxylate production was determined i n reaction mixtures in which the hydroxylamine was