5-Enolpyruvylshikimate-3-phosphate Synthase of Bacillus subtilis is an Allosteric Enzyme. Analysis of Arg24Asp, Pro105Ser and His385Lys mutations suggests a hidden phosphoenol pyruvate-binding site

Abstract

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.

Point mutation of a conserved arginine (104) to lysine introduces hypersensitivity to inhibition by glyphosate in the 5‐enolpyruvylshikimate‐3‐phosphate synthase of Bacillus subtilis

“…Sequence analysis of EPSPs from several bacteria and plants has revealed stretches of conserved amino acid residues [4]. Thus, a critical role in the EPSPs catalytic reaction has been assigned to highly conserved Gly 96, Pro 1°1, His 3s5 and Arg 24 residues [6,16].…”

“…The reaction mechanism, order of binding of substrates, shikimate 3-phosphate and phosphoenol pyruvate and the competitive inhibition of the reaction by glyphosate (Vs PEP) is [17], Bacillus subtilis [12,16] and Petunia hybrida [18]. Therefore, it is logical to presume that the highly conserved residues will represent domains critical for substrate interaction with the enzyme.…”

“…subtilis has been shown to be modulated by monovalent cations [12]. Of several cations tested ammonium ions were shown to maximally activate the enzyme and also sensitize the enzyme to inhibition by glyphosate [12,16]. Wild type enzyme upon activation by ammonia was 25-fold more sensitive to inhibition by glyposate than the unactivated enzyme [12].…”

“…In addition to altered K~ (glyphosate) in R104K, ammonia induced activation of the enzyme activity was abolished completely. The kinetic interaction between PEP, glypho- [16]. A secondary, non-catalytic PEP binding site is located around residue His 385 while the catalytic site is located within residues 90-120.…”

“…A secondary, non-catalytic PEP binding site is located around residue His 385 while the catalytic site is located within residues 90-120. It has been postulated that the interaction of ammonia with the enzyme results in blocking of the non-catalytic PEP binding site resulting in channeling of PEP towards the catalytic site [16]. Ammonia lowers the Km for PEP probably by blocking the non-catalytic PEP binding site and exposing the reactive catalytic site.…”