Isocitrate dehydrogenase and glyoxylate cycle enzyme activities in Bradyrhizobium japonicum under various growth conditi
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© Springer-Verlag 1998
O R I G I N A L PA P E R
Laura S. Green · Dale B. Karr · David W. Emerich
Isocitrate dehydrogenase and glyoxylate cycle enzyme activities in Bradyrhizobium japonicum under various growth conditions
Received: 10 October 1997 / Accepted: 16 January 1998
Abstract Bradyrhizobium japonicum, the nitrogen-fixing symbiotic partner of soybean, was grown on various carbon substrates and assayed for the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase. The highest levels of isocitrate lyase [165–170 nmol min–1 (mg protein)–1] were found in cells grown on acetate or β-hydroxybutyrate, intermediate activity was found after growth on pyruvate or galactose, and very little activity was found in cells grown on arabinose, malate, or glycerol. Malate synthase activity was present in arabinose- and malate-grown cultures and increased by only 50–80% when cells were grown on acetate. B. japonicum bacteroids, harvested at four different nodule ages, showed very little isocitrate lyase activity, implying that a complete glyoxylate cycle is not functional during symbiosis. The apparent Km of isocitrate lyase for D,L-isocitrate was fourfold higher than that of isocitrate dehydrogenase (61.5 and 15.5 µM, respectively) in desalted crude extracts from acetate-grown B. japonicum. When isocitrate lyase was induced, neither the Vmax nor the D,L-isocitrate Km of isocitrate dehydrogenase changed, implying that isocitrate dehydrogenase is not inhibited by covalent modification to facilitate operation of the glyoxylate cycle in B. japonicum. Key words Isocitrate lyase · Malate synthase · Glyoxylate cycle · Bradyrhizobium japonicum · Isocitrate dehydrogenase · Bacteroids
Introduction The glyoxylate cycle, consisting of isocitrate lyase and malate synthase, is an inducible pathway required for the
L. S. Green (Y) · D. B. Karr · D. W. Emerich Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA Tel. +1-573-771-9076; Fax +1-573-882-5635 e-mail: [email protected]
growth of many bacteria on two-carbon substrates such as acetate (Kornberg 1966; Cronan and LaPorte 1996). The cycle bypasses the decarboxylating reactions of the tricarboxylic acid cycle, providing for the net production of succinate from two molecules of acetyl-CoA, and thus supports gluconeogenesis and other biosynthetic processes during growth on two-carbon substrates. However, the tricarboxylic acid cycle decarboxylations catalyzed by isocitrate dehydrogenase and α-ketoglutarate dehydrogenase are important energy-yielding steps, and therefore the relative flux of carbon through these reactions versus the glyoxylate cycle must be regulated according to the relative need of the cell for energy versus biosynthetic precursors. Consequently, isocitrate lyase is generally only induced under conditions in which it is required for growth. Bradyrhizobium japonicum is a soil bacterium that forms a nitrogen-fixing symbiosis with soybean. In its symbiotic (bacteroid) form, B. japonicum relies entirely on the
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