It Takes Two to Tango: Defining an Essential Second Active Site in Pyridoxal 5′-Phosphate Synthase

The prevalent de novo biosynthetic pathway of vitamin B6 involves only two enzymes (Pdx1 and Pdx2) that form an ornate multisubunit complex functioning as a glutamine amidotransferase. The synthase subunit, Pdx1, utilizes ribose 5-phosphate and glyceraldehyde 3-phosphate, as well as ammonia derived from the glutaminase activity of Pdx2 to directly form the cofactor vitamer, pyridoxal 5′-phosphate. Given the fact that a single enzyme performs the majority of the chemistry behind this reaction, a complicated mechanism is anticipated. Recently, the individual steps along the reaction co-ordinate are beginning to be unraveled. In particular, the binding of the pentose substrate and the first steps of the reaction have been elucidated but it is not known if the latter part of the chemistry, involving the triose sugar, takes place in the same or a disparate site. Here, we demonstrate through the use of enzyme assays, enzyme kinetics, and mutagenesis studies that indeed a second site is involved in binding the triose sugar and moreover, is the location of the final vitamin product, pyridoxal 5′-phosphate. Furthermore, we show that product release is triggered by the presence of a PLP-dependent enzyme. Finally, we provide evidence that a single arginine residue of the C terminus of Pdx1 is responsible for coordinating co-operativity in this elaborate protein machinery

Biofuels Feedstock Development Program annual progress report for 1991

This report provides an overview of the ongoing research funded in 1991 by the Department of Energy's Biofuels Feedstock Development Program (BFDP). The BFDP is managed by the Environmental Sciences Division of the Oak Ridge National Laboratory and encompasses the work formerly funded by the Short Rotation Woody Crops Program and the Herbaceous Energy Crops Program. The combined program includes crop development research on both woody and herbaceous energy crop species, cross-cutting energy and environmental analysis and integration, and information management activities. Brief summaries of 26 different program activities are included in the report

The Assembly of the Plasmodial PLP Synthase Complex Follows a Defined Course

Background: Plants, fungi, bacteria and the apicomplexan parasite Plasmodium falciparum are able to synthesize vitamin B6 de novo, whereas mammals depend upon the uptake of this essential nutrient from their diet. The active form of vitamin B6 is pyridoxal 5-phosphate (PLP). For its synthesis two enzymes, Pdx1 and Pdx2, act together, forming a multimeric complex consisting of 12 Pdx1 and 12 Pdx2 protomers. Methodology/Principal Findings: Here we report amino acid residues responsible for stabilization of the structural and enzymatic integrity of the plasmodial PLP synthase, identified by using distinct mutational analysis and biochemical approaches. Residues R85, H88 and E91 (RHE) are located at the Pdx1:Pdx1 interface and play an important role in Pdx1 complex assembly. Mutation of these residues to alanine impedes both Pdx1 activity and Pdx2 binding. Furthermore, changing D26, K83 and K151 (DKK), amino acids from the active site of Pdx1, to alanine obstructs not only enzyme activity but also formation of the complex. In contrast to the monomeric appearance of the RHE mutant, alteration of the DKK residues results in a hexameric assembly, and does not affect Pdx2 binding or its activity. While the modelled position of K151 is distal to the Pdx1:Pdx1 interface, it affects the assembly of hexameric Pdx1 into a functional dodecamer, which is crucial for PLP synthesis. Conclusions/Significance: Taken together, our data suggest that the assembly of a functional Pdx1:Pdx2 complex follows

Light regulation of metabolic pathways in fungi

Light represents a major carrier of information in nature. The molecular machineries translating its electromagnetic energy (photons) into the chemical language of cells transmit vital signals for adjustment of virtually every living organism to its habitat. Fungi react to illumination in various ways, and we found that they initiate considerable adaptations in their metabolic pathways upon growth in light or after perception of a light pulse. Alterations in response to light have predominantly been observed in carotenoid metabolism, polysaccharide and carbohydrate metabolism, fatty acid metabolism, nucleotide and nucleoside metabolism, and in regulation of production of secondary metabolites. Transcription of genes is initiated within minutes, abundance and activity of metabolic enzymes are adjusted, and subsequently, levels of metabolites are altered to cope with the harmful effects of light or to prepare for reproduction, which is dependent on light in many cases. This review aims to give an overview on metabolic pathways impacted by light and to illustrate the physiological significance of light for fungi. We provide a basis for assessment whether a given metabolic pathway might be subject to regulation by light and how these properties can be exploited for improvement of biotechnological processes

Dissection of contributions from invariant amino acids to complex formation and catalysis in the heteromeric pyridoxal 5-phosphate synthase complex from Bacillus subtilis

Pyridoxal 5-phosphate (PLP), an active form of vitamin B(6), is one of the most versatile cofactors and is involved in numerous biochemical reactions. The main pathway for de novo PLP biosynthesis leads to direct formation of PLP from a pentose and triose. This reaction is catalyzed by the heteromeric PLP synthase, consisting of the synthase subunit Pdx1 and the glutaminase subunit Pdx2. l-Glutamine hydrolysis by Pdx2 supplies ammonia to Pdx1 for incorporation into PLP. Autonomous glutaminase Pdx2 is inactive; however, interaction with Pdx1 leads to enzymatic activity. Oxyanion hole formation in the active site of Pdx2 is required for substrate binding and was suggested as the prime event of enzyme activation. Here, we dissect interactions required for complex formation from interactions required for catalytic activation of the glutaminase. The three-dimensional structural analysis suggested a number of invariant residues that regulate complex formation and enzyme activation. We have replaced several of these invariant residues by site-directed mutagenesis in an effort to understand their function. In addition to the biochemical characterization of enzyme activity, the generated protein variants were studied by isothermal calorimetry to investigate their role in complex formation. The assembled data describe a multistep activation mechanism. Residues of helix alphaN of Pdx1 are essential for formation of the Pdx1-Pdx2 complex and also stabilize the oxyanion hole. Thus, these interactions describe the encounter complex. On the other hand, residues at the N-terminal face of the (betaalpha)(8) barrel of Pdx1 contribute to interface formation and are required for the organization of the catalytic center; thus, these interactions describe the Michaelis complex. However, the main players for formation of the Michaelis complex reside on Pdx2, as replacement of residues at the N-terminal face of the (betaalpha)(8) barrel of Pdx1 leads to reduction but not complete inactivation of the glutaminase