Riboflavin (vitamin B 2 ) serves as the precursor for FMN and FAD in almost all organisms that utilize the redox-active isoalloxazine ring system as a coenzyme in enzymatic reactions. The role of flavin, however, is not limited to redox processes, as 10% of flavin-dependent enzymes catalyze nonredox reactions. Moreover, the flavin cofactor is also widely used as a signaling and sensing molecule in biological processes such as phototropism and nitrogen fixation. Here, we present a study of 374 flavin-dependent proteins analyzed with regard to their function, structure and distribution among 22 archaeal, eubacterial, protozoan and eukaryotic genomes. More than 90% of flavin-dependent enzymes are oxidoreductases, and the remaining enzymes are classified as transferases (4.3%), lyases (2.9%), isomerases (1.4%) and ligases (0.4%). The majority of enzymes utilize FAD (75%) rather than FMN (25%), and bind the cofactor noncovalently (90%). High-resolution structures are available for about half of the flavoproteins. FAD-containing proteins predominantly bind the cofactor in a Rossmann fold ( 50%), whereas FMN-containing proteins preferably adopt a (ba) 8 -(TIM)-barrel-like or flavodoxin-like fold. The number of genes encoding flavin-dependent proteins varies greatly in the genomes analyzed, and covers a range from 0.1% to 3.5% of the predicted genes. It appears that some species depend heavily on flavin-dependent oxidoreductases for degradation or biosynthesis, whereas others have minimized their flavoprotein arsenal. An understanding of 'flavin-intensive' lifestyles, such as in the human pathogen Mycobacterium tuberculosis, may result in valuable new intervention strategies that target either riboflavin biosynthesis or uptake.

Our quest to understand the complex inner workings of the cell depends on the development of new technologies that allow the study of global regulatory events as they happen within their native cellular environment. Post-translational processing of proteins by proteases is one such regulatory process that can control many aspects of basic cell biology. In this issue of the Biochemical Journal, Timmer et al. describe a new proteomic approach that can be used to globally monitor constitutive proteolytic events in vivo. Using bacterial, human, yeast and mouse cells, the authors show that this methodology provides a comprehensive map of constitutive trimming events mediated by regulatory proteases such as methionine aminopeptidase. This study also identifies previously uncharacterized processing events that highlight potential novel regulatory mechanisms mediated by proteolysis.

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...