5-Enolpyruvylshikimate-3-phosphate synthase of Bacillus subtilis has been cloned, expressed and purified to near homogeneity. Clustal alignment of the amino acid sequences from different bacteria revealed several conserved residues located in the N-terminal, middle and C-terminal domains. The role of conserved Arg24, Pro105, and His385 residues has been examined by site-directed mutagenesis. Steady-state kinetic analysis of the native synthase exhibited allosteric behaviour, a feature thought to be unique amongst bacterial and plant 5-enolpyruvylshikimate-3-phosphate synthase enzymes investigated so far. Both substrates, phosphoenolpyruvate (P-pyruvate) and shikimate 3-phosphate have multiple interaction sites. There are two sites for P-pyruvate binding, catalytic and non-catalytic. Glyphosate (N-phosphonomethyl glycine) competes for binding at the catalytic site and does not interact at the secondary site. Glyphosate in the absence of ammonium ions increases cooperativity of P-pyruvate binding and favors dimerization of the enzyme through an interaction between P-pyruvate-binding sites. The ammonium-ion-activated 5-enolpyruvylshikimate-3-phosphate synthase displays no cooperativity with respect to P-pyruvate. Absence of ammonium ions decreases affinity for substrates and introduces cooperativity. Cooperativity was also introduced in the enzyme by point mutations, Arg24-->Asp and His385-->Lys. The latter mutant of the native enzyme exists as a dimer and aggregates to a tetrameric form in the presence of glyphosate. The occurrence of multimeric forms of the synthase has been demonstrated by staining for the enzyme activity on the native gel and by resolving purified enzyme preparations on a sucrose density gradient. A model describing the alteration in the aggregation status of the enzyme by the inhibitor, activator and the substrates has been proposed.

The role of a conserved arginine (R104) in the putative phosphoenol pyruvate binding region of 5-enolpyruvyl shikimate-3-phosphate synthase of Bacillus subtilis has been investigated. Employing site directed mutagenesis arginine was substituted by lysine or glutamine. Native and mutant proteins were expressed and purified to near homogeneity. Estimation of Michaelis and inhibitor constants of the native and mutant proteins exhibited altered substrate-inhibitor binding mode and constants. Mutation RI04K hypersensitized the enzyme reaction to inhibition by glyphosate. The role of R104 in discriminating between glyphosate and phosphoenol pyruvate is discussed.

5‐Enol pyruvylshikimate‐3‐phosphate synthase of Bacillus subtilis has been cloned, expressed and purified to near homogeneity. Clustal alignment of the amino acid sequences from different bacteria revealed several conserved residues located in the N‐terminal, middle and C‐terminal domains. The role of conserved Arg24, Pro105, and His385 residues has been examined by site‐directed mutagenesis. Steady‐state kinetic analysis of the native synthase exhibited allosteric behaviour, a feature thought to be unique amongst bacterial and plant 5‐enol pynivylshikimate‐3‐phosphate synthase enzymes investigated so far. Both substrates, phosphoenol pyruvate (P ‐pyruvate) and shikimate 3‐phosphate have multiple interaction sites. There are two sites for P ‐pyruvate binding, catalytic and non‐catalytic. Glyphosate (N ‐phosphonomethyl glycine) competes for binding at the catalytic site and does not interact at the secondary site. Glyphosate in the absence of ammonium ions increases cooperativity of P ‐pyruvate binding and favors dimerization of the enzyme through an interaction between P ‐pyruvate‐binding sites. The ammonium‐ion‐activated 5‐enol pyruvylshikimate‐3‐phosphate synthase displays no cooperativity with respect to P‐pyruvate. Absence of ammonium ions decreases affinity for substrates and introduces cooperativity. Cooperativity was also introduced in the enzyme by point mutations, Arg24→Asp and His385→Lys. The latter mutant of the native enzyme exists as a dimer and aggregates to a tetrameric form in the presence of glyphosate. The occurrence of multimeric forms of the synthase has been demonstrated by staining for the enzyme activity on the native gel and by resolving purified enzyme preparations on a sucrose density gradient. A model describing the alteration in the aggregation status of the enzyme by the inhibitor, activator and the substrates has been proposed.

Giant cells associated with egg-laying females of Meloidogyne javanica in lima bean, Phaseolus lunatus cv. L-136, were examined by light and electron microscopy. These giant cells have characteristics that are typical of nematode-induced giant cells in a wide range of hosts, but they differ in that they (i) are less closely associated with xylem vessels, (ii) contain a very large number of plastids which are devoid of starch grains, and (iii) contain several different forms of cytoplasmic crystals. One form of the crystal is associated with a large number of "spiny" vesicles. The possible role of these features, especially the crystals, in the giant cell response of lima bean is discussed.

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