Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis.

Substrate channeling has emerged as a common mechanism for enzymatic intermediate transfer. A conspicuous gap in knowledge concerns the use of covalent lysine imines in the transfer of carbonyl-group-containing intermediates, despite their wideuse in enzymatic catalysis. Here we show how imine chemistry operates in the transfer of covalent intermediates in pyridoxal 5'-phosphate biosynthesis by the Arabidopsis thaliana enzyme Pdx1. An initial ribose 5-phosphate lysine imine is converted to the chromophoric I320 intermediate, simultaneously bound to two lysine residues and partially vacating the active site, which creates space for glyceraldehyde 3-phosphate to bind. Crystal structures show how substrate binding, catalysis and shuttling are coupled to conformational changes around strand ß6 of the Pdx1 (ßα)8-barrel. The dual-specificity active site and imine relay mechanism for migration of carbonyl intermediates provide elegant solutions to the challenge of coordinating a complex sequence of reactions that follow a path of over 20 Å between substrate- and product-binding sites.

Molecular modeling of family GH16 glycoside hydrolases: potential roles for xyloglucan transglucosylases/hydrolases in cell wall modification in the poaceae.

Family GH16 glycoside hydrolases can be assigned to five subgroups according to their substrate specificities, including xyloglucan transglucosylases/hydrolases (XTHs), (1,3)-beta-galactanases, (1,4)-beta-galactanases/kappa-carrageenases, "nonspecific" (1,3/1,3;1,4)-beta-D-glucan endohydrolases, and (1,3;1,4)-beta-D-glucan endohydrolases. A structured family GH16 glycoside hydrolase database has been constructed (http://www.ghdb.uni-stuttgart.de) and provides multiple sequence alignments with functionally annotated amino acid residues and phylogenetic trees. The database has been used for homology modeling of seven glycoside hydrolases from the GH16 family with various substrate specificities, based on structural coordinates for (1,3;1,4)-beta-D-glucan endohydrolases and a kappa-carrageenase. In combination with multiple sequence alignments, the models predict the three-dimensional (3D) dispositions of amino acid residues in the substrate-binding and catalytic sites of XTHs and (1,3/1,3;1,4)-beta-d-glucan endohydrolases; there is no structural information available in the databases for the latter group of enzymes. Models of the XTHs, compared with the recently determined structure of a Populus tremulos x tremuloides XTH, reveal similarities with the active sites of family GH11 (1,4)-beta-D-xylan endohydrolases. From a biological viewpoint, the classification, molecular modeling and a new 3D structure of the P. tremulos x tremuloides XTH establish structural and evolutionary connections between XTHs, (1,3;1,4)-beta-D-glucan endohydrolases and xylan endohydrolases. These findings raise the possibility that XTHs from higher plants could be active not only on cell wall xyloglucans, but also on (1,3;1,4)-beta-D-glucans and arabinoxylans, which are major components of walls in grasses. A role for XTHs in (1,3;1,4)-beta-D-glucan and arabinoxylan modification would be consistent with the apparent overrepresentation of XTH sequences in cereal expressed sequence tags databases.

X-ray crystal structure of Saccharomyces cerevisiae Pdx1 provides insights into the oligomeric nature of PLP synthases.

The universal enzymatic cofactor vitamin B6 can be synthesized as pyridoxal 5-phosphate (PLP) by the glutamine amidotransferase Pdx1. We show that Saccharomyces cerevisiae Pdx1 is hexameric by analytical ultracentrifugation and by crystallographic 3D structure determination. Bacterial homologues were previously reported to exist in hexamer:dodecamer equilibrium. A small sequence insertion found in yeast Pdx1 elevates the dodecamer dissociation constant when introduced into Bacillus subtilis Pdx1. Further, we demonstrate that the yeast Pdx1 C-terminus contacts an adjacent subunit, and deletion of this segment decreases enzymatic activity 3.5-fold, suggesting a role in catalysis.

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.

Structural and thermodynamic insights into the assembly of the heteromeric pyridoxal phosphate synthase from Plasmodium falciparum.

Pyridoxal 5'-phosphate (PLP) is required as a cofactor by many enzymes. The predominant de novo biosynthetic route is catalyzed by a heteromeric glutamine amidotransferase consisting of the synthase subunit Pdx1 and the glutaminase subunit Pdx2. Previously, Bacillus subtilis PLP synthase was studied by X-ray crystallography and complex assembly had been characterized by isothermal titration calorimetry. The fully assembled PLP synthase complex contains 12 individual Pdx1/Pdx2 glutamine amidotransferase heterodimers. These studies revealed the occurrence of an encounter complex that is tightened in the Michaelis complex when the substrate l-glutamine binds. In this study, we have characterized complex formation of PLP synthase from the malaria-causing human pathogen Plasmodium falciparum using isothermal titration calorimetry. The presence of l-glutamine increases the tightness of the interaction about 30-fold and alters the thermodynamic signature of complex formation. The thermodynamic data are integrated in a 3D homology model of P. falciparum PLP synthase. The negative experimental heat capacity (C(p)) describes a protein interface that is dominated by hydrophobic interactions. In the absence of l-glutamine, the experimental C(p) is less negative than in its presence, contrasting to the previously characterised bacterial PLP synthase. Thus, while the encounter complexes differ, the Michaelis complexes of plasmodial and bacterial systems have similar characteristics concerning the relative contribution of apolar/polar surface area. In addition, we have verified the role of the N-terminal region of PfPdx1 for complex formation. A "swap mutant" in which the complete alphaN-helix of plasmodial Pdx1 was exchanged with the corresponding segment from B. subtilis shows cross-binding to B. subtilis Pdx2. The swap mutant also partially elicits glutaminase activity in BsPdx2, demonstrating that formation of the protein complex interface via alphaN and catalytic activation of the glutaminase are linked processes.

Structure of a bacterial pyridoxal 5'-phosphate synthase complex.

Vitamin B6 is an essential metabolic cofactor that has more functions in humans than any other single nutrient. Its de novo biosynthesis occurs through two mutually exclusive pathways that are absent in animals. The predominant pathway found in most prokaryotes, fungi, and plants has only recently been discovered. It is distinguished by a glutamine amidotransferase, which is remarkable in that it alone can synthesize the cofactor form, pyridoxal 5'-phosphate (PLP), directly from a triose and a pentose saccharide and glutamine. Here we report the 3D structure of the PLP synthase complex with substrate glutamine bound as well as those of the individual synthase and glutaminase subunits Pdx1 and Pdx2, respectively. The complex is made up of 24 protein units assembled like a cogwheel, a dodecameric Pdx1 to which 12 Pdx2 subunits attach. In contrast to the architecture of previously determined glutamine amidotransferases, macromolecular assembly is directed by an N-terminal alpha-helix on the synthase. Interaction with the synthase subunit leads to glutaminase activation, resulting in formation of an oxyanion hole, a prerequisite for catalysis. Mutagenesis permitted identification of the remote glutaminase and synthase catalytic centers and led us to propose a mechanism whereby ammonia shuttles between these active sites through a methionine-rich hydrophobic tunnel.