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Bradyrhizobium japonicum NADP(+)-dependent isocitrate dehydrogenase was purified both from cultured cells and from the symbiotic form of the bacteria and was found to be identical in terms of N-terminal amino acid sequence, kinetics, and physicochemical properties. Magnesium and glycerol were absolute requirements for maintaining enzyme activity. The N-terminal amino acid sequence of the enzyme was more similar to the sequences from soybean and yeast than to other bacterial sequences. There was no immunological cross-reaction of antibodies from B. japonicum isocitrate dehydrogenase to extracts of soybean, pea, or Escherichia coli, but there was detectable, although weak, cross-reaction of antibodies from E. coli with the B. japonicum enzyme. B. japonicum isocitrate dehydrogenase displayed strong inhibition by NADH, indicating that during symbiotic nitrogen fixation the enzyme activity would be markedly reduced in planta. The enzyme displayed a calcium-dependent hysteresis, with a pronounced lag lasting as long as 2 min. Hysteresis was evident at concentrations of magnesium less than 0.5 mM and calcium greater than 1 microM. The hysteresis could be alleviated by excess magnesium or by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. The results suggest two roles for magnesium during catalysis; one magnesium may be needed to convert the enzyme into the steady-state form and the second needed for chelation of isocitrate for catalysis. The calcium-dependent hysteretic behavior of B. japonicum NADP(+)-isocitrate dehydrogenase suggested that this metal could serve as an intracellular regulator during symbiosis., (Copyright 2000 Academic Press.)

Alanine dehydrogenase (ALADH) from soybean nodule bacteriods was purified 184-fold with 14% yield, using ammonium sulfate precipitation, hydroxylapatite, gel filtration, ion exchange, and dye affinity chromatography. The subunit molecular weight was 43,000 and the native molecular weight was approximately 190,000, suggesting that ALADH is a tetramer. ALADH was confined to the bacteroid cytosol fraction only. ALADH is specific for NAD(H) and does not use NADP(H) as a substrate, but it does use glyoxylate and hydroxypyruvate as substrates in lieu of pyruvate. The pH optimum was 8.5 for the amination reaction and 10.0 for the deamination reaction. The apparent Michaelis constants for NADH, NH4+, pyruvate, L-alanine, and NAD were 86 microM, 8.9 mM, 0.49 mM, 1 mM and 200 microM, respectively. High concentrations of pyruvate, L-alanine, or NH4+ caused inhibition of activity with Ki's of 8.6 mM, 6.5-15 mM, and 188 mM, respectively. The amination reaction of ALADH was 95-100% of the control at levels of NADH/NAD corresponding to those measured in isolated bacteroids. The deamination reaction, on the other hand, was only 35-40% of control. Thus, an aminating role for ALADH is possible.

A number of fluorescence studies, both of trp residues and bound NADH, have been reported for porcine malate dehydrogenase (MDH). The large number of trp residues (six) complicates the interpretation of some studies. To circumvent this we have performed studies with a two-tryptophan (per subunit) MDH from Bradyrhizobium japonicum 3I1B-143 bacteroids. We have performed phase/modulation fluorescence lifetime measurements, as a function of temperature and added quencher KI, in order to resolved the 1.2-ns (blue) and 6.5-ns (red) contributions from the two classes of trp residues. Anisotropy decay studies have also been performed. The binding of NADH dynamically quenches the fluorescence of both trp residues, but, unlike mammalian cytoplasmic and mitochondrial MDH, there is not a large enhancement in fluorescence of bound NADH upon forming a ternary complex with either tartronic acid or D-malate.

Acetoacetyl-CoA thiolase of Bradyrhizobium japonicum bacteroids has been purified greater than 130-fold. The enzyme has a molecular weight of 180,000 +/- 15,000 and consists of four identical subunits of 44,000 +/- 2,000. The enzyme was specific for acetoacetyl-CoA; ketodecanoyl-CoA did not serve as a substrate. Catalysis proceeds via a ping-pong mechanism. Iodoacetamide effectively inhibited the enzyme but acetoacetyl-CoA provided considerable protection against this compound. Magnesium was found to inhibit both the thiolysis reaction and the condensation reaction. Acetoacetyl-CoA thiolysis activity was not affected by potassium, ammonium, or several organic acids but was found to be inhibited by NADH. The inhibition by NADH may have an effect during the decline of the symbiosis.