In prokaryotes, cognate toxin-antitoxin pairs have long been known, but no three-dimensional structure has been available for any given complex from Mycobacterium tuberculosis. Here we report the crystal structure and activity of a member of the VapBC family of complexes from M. tuberculosis. The toxin VapC-5 is a compact, 150 residues, two domain ␣/␤ protein. Bent around the toxin is the VapB-5 antitoxin, a 33-residue ␣-helix. Assays suggest that the toxin is an Mg-enabled endoribonuclease, inhibited by the antitoxin. The lack of DNase activity is consistent with earlier suggestions that the complex represses its own operon. Furthermore, analysis of the interactions in the binding of the antitoxin to the toxin suggest that exquisite control is required to protect the bacteria cell from toxic VapC-5.

4-Diphosphocytidyl-2C-methyl-D-erythritol kinase, an essential enzyme in the nonmevalonate pathway of isopentenyl diphosphate and dimethylallyl diphosphate biosynthesis, catalyzes the single ATP-dependent phosphorylation stage affording 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate. The 2-Å resolution crystal structure of the Escherichia coli enzyme in a ternary complex with substrate and a nonhydrolyzable ATP analogue reveals the molecular determinants of specificity and catalysis. The enzyme subunit displays the ␣͞␤ fold characteristic of the galactose kinase͞ homoserine kinase͞mevalonate kinase͞phosphomevalonate kinase superfamily, arranged into cofactor and substrate-binding domains with the catalytic center positioned in a deep cleft between domains. Comparisons with related members of this superfamily indicate that the core regions of each domain are conserved, whereas there are significant differences in the substrate-binding pockets. The nonmevalonate pathway is essential in many microbial pathogens and distinct from the mevalonate pathway used by mammals. The high degree of sequence conservation of the enzyme across bacterial species suggests similarities in structure, specificity, and mechanism. Our model therefore provides an accurate template to facilitate the structure-based design of broad-spectrum antimicrobial agents.galactose kinase͞homoserine kinase͞mevalonate kinase͞ phosphomevalonate kinase ͉ enzyme mechanism ͉ nonmevalonate ͉ phosphorylation T he AT P-dependent 4-diphosphocytidyl-2C-methyl-Derythritol (CDP-ME) kinase (EC 2.7.1.148) participates in the biosynthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). These isomers are the universal five-carbon precursors of isoprenoids, a diverse and important family of natural products that includes sterols, dolichols, triterpenes, and ubiquinones, and components of macromolecules such as the prenyl groups of prenylated proteins and isopentenylated tRNAs (1-3). Isoprenoids contribute to many biological functions, including electron transport in respiration and photosynthesis, hormone-based signaling, apoptosis, meiosis, protein cleavage, and degradation (4). In addition, they provide important structural components of cell membranes (1).Two biosynthetic routes to IPP and DMAPP have evolved. In eukaryotes, archaebacteria, and a few eubacteria, the precursor biosynthesis is through the mevalonate pathway (3-7). This begins with the conversion of three molecules of acetyl-CoA to 3-hydroxy-3-methylglutaryl-CoA followed by reduction, phosphorylation, and decarboxylation to generate IPP, some of which is isomerized to DMAPP. The last three steps in this pathway are ATP-dependent and catalyzed by the structurally related mevalonate kinase (MVK), phosphomevalonate kinase, and mevalonate 5-diphosphate decarboxylase.In chloroplasts, algae, cyanobacteria, most eubacteria, and the apicomplexa, IPP and DMAPP synthesis is accomplished by seven enzymes in a pathway named after one of the intermediates, the 1-deoxy-D-xylulose-5-ph...

The ®rst results of a medium-scale structural genomics program clearly demonstrate the value of using a mediumthroughput crystallization approach based on a two-step procedure: a large screening step employing robotics, followed by manual or automated optimization of the crystallization conditions. The structural genomics program was based on cloning in the Gateway 2 vectors pDEST17, introducing a long 21-residue tail at the N-terminus. So far, this tail has not appeared to hamper crystallization. In ten months, 25 proteins were subjected to crystallization; 13 yielded crystals, of which ten led to usable data sets and ®ve to structures. Furthermore, the results using a robot dispensing 50±200 nl drops indicate that smaller protein samples can be used for crystallization. These still partial results might indicate present and future directions for those who have to make crucial choices concerning their crystallization platform in structural genomics programs.