Thermodynamic Characterization of the Protein−Protein Interaction in the Heteromeric Bacillus subtilis Pyridoxalphosphate Synthase

Publisher Website
Google Scholar
Abstract
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 μM in its absence and presence, respectively (at 25 °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 (ΔCp) agree with a protein interface governed by hydrophobic interactions. The measured heat capacity change is also a function of l-glutamine, with a negative ΔCp 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.