Thermodynamic characterization of the protein-protein interaction in the heteromeric Bacillus subtilis pyridoxalphosphate synthase.

Two biosynthetic routes for the synthesis of pyridoxal 5'-phosphate (PLP), the biologically active compound of vitamin B6, have been characterized. The major pathway leads to direct formation of PLP from a pentasaccharide and a trisaccharide and is operative in plants, fungi, protozoa, and bacteria. This reaction is catalyzed by a single glutamine amidotransferase enzyme complex consisting of a pyridoxal synthase, termed Pdx1, and a glutaminase, termed Pdx2. In this complex, Pdx2 generates ammonia from L-glutamine and supplies it to Pdx1 for incorporation into PLP. The glutaminase activity of Pdx2 requires the presence of Pdx1 in a heteromeric complex, previously characterized by a crystallographic three-dimensional (3D) structure determination. Here, we give a thermodynamic description of complex formation of Bacillus subtilis PLP synthase in the absence or presence of L-glutamine. We show that L-glutamine directly affects the tightness of the protein complex, which exhibits dissociation constants of 6.9 and 0.3 microM in its absence and presence, respectively (at 25 degrees C). This result relates to the positioning of L-glutamine on the heterodimer interface as seen in the 3D structure. In an analysis of the temperature dependence of the enthalpy, negative heat capacity changes (deltaCp) agree with a protein interface governed by hydrophobic interactions. The measured heat capacity change is also a function of L-glutamine, with a negative deltaCp in the presence of L-glutamine and a more negative one in its absence. These findings suggest that L-glutamine not only affects the strength of complex formation but also determines the forces involved in complex formation, with regard to different relative contributions of hydrophobic and hydrophilic interactions.