Vitamin B6 is one of nature's most versatile cofactors. Most organisms synthesize vitamin B6 via a recently discovered pathway employing the proteins Pdx1 and Pdx2. Here we present an in-depth characterization of the respective orthologs from the malaria parasite, Plasmodium falciparum. Expression profiling of Pdx1 and -2 shows that blood-stage parasites indeed possess a functional vitamin B6 de novo biosynthesis. Recombinant Pdx1 and Pdx2 form a complex that functions as a glutamine amidotransferase with Pdx2 as the glutaminase and Pdx1 as pyridoxal-5-phosphate synthase domain. Complex formation is required for catalytic activity of either domain. Pdx1 forms a chimeric bi-enzyme with the bacterial YaaE, a Pdx2 ortholog, both in vivo and in vitro, although this chimera does not attain full catalytic activity, emphasizing that species-specific structural features govern the interaction between the protein partners of the PLP synthase complexes in different organisms. To gain insight into the activation mechanism of the parasite bi-enzyme complex, the three-dimensional structure of Pdx2 was determined at 1.62 Å . The obstruction of the oxyanion hole indicates that Pdx2 is in a resting state and that activation occurs upon Pdx1-Pdx2 complex formation.Plasmodium falciparum is the causative agent of severe malaria. Each year up to two million human deaths and enormous economic losses are attributed to this parasite. Drug resistance in P. falciparum has been aggravating the problem in many parts of the world during the last two decades, which considering the lack of a protective vaccine, is the major obstacle to combat the disease. Hence, new antimalarials are urgently needed. Requirements for nutrients and vitamins have previously been discussed as possible novel targets (1). Indeed the P. falciparum genome contains genes that encode enzymes necessary for the syntheses of the vitamin precursor chorismate (2-4), vitamin B6 (5, 6), and the vitaminlike cofactor lipoic acid (7).Vitamin B6 is renowned in the medical field as being involved in more bodily functions than any other single nutrient. It is required for the maintenance of physical as well as mental health. The term "vitamin B6" collectively refers to the vitamers pyridoxal, pyridoxine, and pyridoxamine, and their respective phosphate esters. The metabolically active form is pyridoxal 5Ј-phosphate (PLP), 6 an essential co-enzyme in numerous pathways such as amino acid metabolism and the biosynthesis of antibiotic compounds. In contrast to mammals, which have to take up vitamin B6 from their diet, bacteria, fungi, plants, and the protozoan P. falciparum have the ability to synthesize the vitamin de novo.Analyses of a number of available genomes has demonstrated that most organisms, including all archaea, fungi, plants, and protozoa and most eubacteria use a class I glutamine amidotransferase (GATase) composed of two domains, a glutaminase and its associated acceptor/ synthase domain to generate vitamin B6 (8 -13). Structural knowledge on class I GATases in genera...

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.

BackgroundSustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes.Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic) proteins due to several advantages.ResultsIn this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes we could develop strains capable of secreting gram quantities of enzyme per liter in fed-batch cultivations. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data.ConclusionIn our experiments we could clearly show the importance of gene optimization and strain characterization for successfully improving secretion levels. We also present a basic guideline how to correctly interpret the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris.

Lipid oxidation is now thought to be an initiating and sustaining event in atherogenesis. Oxidatively fragmented phospholipids, namely 1-palmitoyl-2-glutaroylsn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), present in minimally modified LDL and atherosclerotic lesions, have been reported to elicit a wide range of pathophysiological responses in the cells of the vascular wall. Nevertheless, the question of their potential sites of action and their primary molecular targets remains open. To address this issue, a series of fluorescently labeled analogs, which differ with regard to structure and binding site of the fluorophore, were synthesized and used as tools for studying the uptake, intracellular stability, and distribution of PGPC and POVPC in vascular smooth muscle cells (VSMCs). We demonstrate that in accordance with their lysophospholipid-like structure, these highly similar molecules transferred rapidly either from aqueous phospholipid dispersions or preloaded native LDL into VSMCs, producing disparate fluorescence patterns irrespective of the attached fluorophore. PGPC derivatives were translocated to the lysosomes. In sharp contrast, POVPC analogs were initially captured in the plasma membrane, most likely in consequence of the formation of covalent adducts with free amino and sulfhydryl groups of proteins and phospholipids.