The Bacillus subtilis Sfp protein activates the peptidyl carrier protein (PCP) domains of surfactin synthetase by transferring the 4Ј-phosphopantetheinyl moiety of coenzyme A (CoA) to a serine residue conserved in all PCPs. Its wide PCP substrate spectrum renders Sfp a biotechnologically valuable enzyme for use in combinatorial non-ribosomal peptide synthesis. The structure of the Sfp-CoA complex determined at 1.8 Å resolution reveals a novel α/β-fold exhibiting an unexpected intramolecular 2-fold pseudosymmetry. This suggests a similar fold and dimerization mode for the homodimeric phosphopantetheinyl transferases such as acyl carrier protein synthase. The active site of Sfp accommodates a magnesium ion, which is complexed by the CoA pyrophosphate, the side chains of three acidic amino acids and one water molecule. CoA is bound in a fashion that differs in many aspects from all known CoA-protein complex structures. The structure reveals regions likely to be involved in the interaction with the PCP substrate.

tRNA‐guanine transglycosylases (TGT) are enzymes involved in the modification of the anticodon of tRNAs specific for Asn, Asp, His and Tyr, leading to the replacement of guanine‐34 at the wobble position by the hypermodified base queuine. In prokaryotes TGT catalyzes the exchange of guanine‐34 with the queuine (.)precursor 7‐aminomethyl‐7‐deazaguanine (preQ1). The crystal structure of TGT from Zymomonas mobilis was solved by multiple isomorphous replacement and refined to a crystallographic R‐factor of 19% at 1.85 angstrom resolution. The structure consists of an irregular (beta/alpha)8‐barrel with a tightly attached C‐terminal zinc‐containing subdomain. The packing of the subdomain against the barrel is mediated by an alpha‐helix, located close to the C‐terminus, which displaces the eighth helix of the barrel. The structure of TGT in complex with preQ1 suggests a binding mode for tRNA where the phosphate backbone interacts with the zinc subdomain and the U33G34U35 sequence is recognized by the barrel. This model for tRNA binding is consistent with a base exchange mechanism involving a covalent tRNA‐enzyme intermediate. This structure is the first example of a (beta/alpha)‐barrel protein interacting specifically with a nucleic acid.

We have solved the 2.5-Å crystal structure of 1-deoxy-D-xylulose-5-phosphate reductoisomerase, an enzyme involved in the mevalonate-independent 2-C-methyl-Derythritol-4-phosphate pathway of isoprenoid biosynthesis. The structure reveals that the enzyme is present as a homodimer. Each monomer displays a V-like shape and is composed of an amino-terminal dinucleotide binding domain, a connective domain, and a carboxyl-terminal four-helix bundle domain. The connective domain is responsible for dimerization and harbors most of the active site. The strictly conserved acidic residues Asp 150 , Glu 152 , Glu 231 , and Glu 234 are clustered at the putative active site and are probably involved in the binding of divalent cations mandatory for enzyme activity. The connective and four-helix bundle domains show significant mobility upon superposition of the dinucleotide binding domains of the three conformational states present in the asymmetric unit of the crystal. A still more pronounced flexibility is observed for a loop spanning residues 186 to 216, which adopts two completely different conformations within the three protein conformers. A possible involvement of this loop in an induced fit during substrate binding is discussed. Isoprenoids, formed by the condensation of varying numbers of isopentenyl diphosphate (IPP)1 units, constitute a major class of both primary and secondary metabolites including, for example, the ubiquitous sterols as well as dolichols, plastochinones, carotenoids, the prenyl side chains of chlorophylls, and ubiquinones (1). In archaea, fungi, and mammals, the central building block of isoprenoids, IPP, is formed from acetyl-CoA via the classical mevalonate pathway that was described in the 1950s (reviewed in Ref. 2). Only about 8 years ago, however, the existence of an alternative IPP biosynthesis pathway was established (3) (Fig. 1). The first step, in which the condensation of pyruvate and D-glyceraldehyde-3-phosphate leads to the formation of 1-deoxy-D-xylulose-5-phosphate (DOXP) under release of CO 2 , is catalyzed by DOXP synthase. Subsequently, DOXP reductoisomerase mediates an intramolecular rearrangement followed by reduction using NADPH as hydrogen donor with 2-C-methyl-D-erythritol-4-phosphate (MEP) as product. In addition to NADPH, this reaction depends on the presence of divalent cations, with Mn 2ϩ being most effective (4). In subsequent steps, MEP is cytidylated by the enzyme CDP-ME synthetase under release of pyrophosphate and phosphorylated by CDP-ME kinase to yield 4-diphosphocytidyl-2-Cmethyl-D-erythritol-2-phosphate (CDP-ME-2-phosphate). This intermediate is then cyclized by MECDP synthase under release of CMP, resulting in the formation of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MECDP) (reviewed in Ref. 5). The remaining terminal steps necessary for the synthesis of IPP are largely unknown as yet, although two genes controlling these steps, gcpE and lytB, have been characterized (6, 7). This second IPP biosynthesis pathway has been named the MEP pathway after its key meta...

Transfer RNA-guanine transglycosylases (TGTs) are evolutionarily ancient enzymes, present in all kingdoms of life, catalyzing guanine exchange within their cognate tRNAs by modified 7-deazaguanine bases. Although distinct bases are incorporated into tRNA at different positions in a kingdom-specific manner, the catalytic subunits of TGTs are structurally well conserved. This review provides insight into the sequential steps along the reaction pathway, substrate specificity, and conformational adaptions of the binding pockets by comparison of TGT crystal structures in complex with RNA substrates of a eubacterial and an archaebacterial species. Substrate-binding modes indicate an evolutionarily conserved base-exchange mechanism with a conserved aspartate serving as a nucleophile through covalent binding to C1' of the guanosine ribose moiety in an intermediate state. A second conserved aspartate seems to control the spatial rearrangement of the ribose ring along the reaction pathway and supposedly operates as a general acid/base. Water molecules inside the binding pocket accommodating interaction sites subsequently occupied by polar atoms of substrates help to elucidate substrate-recognition and substrate-specificity features. This emphasizes the role of water molecules as general probes to map binding-site properties for structure-based drug design. Additionally, substrate-bound crystal structures allow the extraction of valuable information about the classification of the TGT superfamily into a subdivision of presumably homologous superfamilies adopting the triose-phosphate isomerase type barrel fold with a standard phosphate-binding motif.

Eubacterial tRNA-guanine transglycosylase (TGT) is involved in the hypermodification of cognate tRNAs, leading to the exchange of G34 by preQ1 at the wobble position in the anticodon loop. Mutation of the tgt gene in Shigella flexneri results in a significant loss of pathogenicity of the bacterium due to inefficient translation of a virulence protein mRNA. Herein, we describe the discovery of a ligand with an unexpected binding mode. On the basis of this binding mode, three slightly deviating pharmacophore hypotheses have been derived. Virtual screening based on this composite pharmacophore model retrieved a set of potential TGT inhibitors belonging to several compound classes. All nine tested inhibitors being representatives of these classes showed activity in the micromolar range, two of them even in the submicromolar range.