The leucine biosynthetic pathway is essential for the growth of Mycobacterium tuberculosis and is a potential target for the design of new anti-tuberculosis drugs. The crystal structure of ␣-isopropylmalate synthase, which catalyzes the first committed step in this pathway, has been determined by multiwavelength anomalous dispersion methods and refined at 2.0-Å resolution in complex with its substrate ␣-ketoisovalerate. The structure reveals a tightly associated, domain-swapped dimer in which each monomer comprises an (␣͞␤)8 TIM barrel catalytic domain, a helical linker domain, and a regulatory domain of novel fold. Mutational and crystallographic data indicate the latter as the site for leucine feedback inhibition of activity. Domain swapping enables the linker domain of one monomer to sit over the catalytic domain of the other, inserting residues into the active site that may be important in catalysis. The ␣-ketoisovalerate substrate binds to an active site zinc ion, adjacent to a cavity that can accommodate acetyl-CoA. Sequence and structural similarities point to a catalytic mechanism similar to that of malate synthase and an evolutionary relationship with an aldolase that catalyzes the reverse reaction on a similar substrate.

The first crystal structure of a drug (daunomycin) bound to a parallel-stranded intermolecular telomeric G4 quadruplex (d(TGGGGT)4) has been determined to high resolution. A planar assemblage of three daunomycin molecules stacks onto the 5' end of the G4 column, with the daunosamine substituents occupying three of the four quadruplex grooves. The surface area of the terminal G-quartet in this parallel DNA quadruplex, presently occupied by three daunomycins, is sufficiently large that it could easily accommodate other potential telomerase inhibitors such as substituted porphyrins or telomestatin.

The modified flavin coenzyme F 420 is found in a restricted number of microorganisms. It is widely distributed in mycobacteria, however, where it is important in energy metabolism, and in Mycobacterium tuberculosis (Mtb) is implicated in redox processes related to non-replicating persistence. In Mtb, the F 420 -dependent glucose-6-phosphate dehydrogenase FGD1 provides reduced F 420 for the in vivo activation of the nitroimidazopyran prodrug PA-824, currently being developed for anti-tuberculosis therapy against both replicating and persistent bacteria. The structure of M. tuberculosis FGD1 has been determined by x-ray crystallography both in its apo state and in complex with F 420 and citrate at resolutions of 1.90 and 1.95 Å , respectively. The structure reveals a highly specific F 420 binding mode, which is shared with several other F 420 -dependent enzymes. Citrate occupies the substrate binding pocket adjacent to F 420 and is shown to be a competitive inhibitor (IC 50 43 M). Modeling of the binding of the glucose 6-phosphate (G6P) substrate identifies a positively charged phosphate binding pocket and shows that G6P, like citrate, packs against the isoalloxazine moiety of F 420 and helps promote a butterfly bend conformation that facilitates F 420 reduction and catalysis.

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Phosphorylation is critical to regulation of the eukaryotic cell cycle. Entry to mitosis is triggered by the cyclin-dependent kinase CDK1 (Cdc2), which is inactivated during the preceding S and G2 phases by phosphorylation of T14 and Y15. Two homologous kinases, Wee1, which phosphorylates Y15, and Myt1, which phosphorylates both T14 and Y15, mediate this inactivation. We have determined the crystal structure of the catalytic domain of human somatic Wee1 (Wee1A) complexed with an active-site inhibitor at 1.8 A resolution. Although Wee1A is functionally a tyrosine kinase, in sequence and structure it most closely resembles serine/threonine kinases such as Chk1 and cAMP kinases. The crystal structure shows that although the catalytic site closely resembles that of other protein kinases, the activation segment contains Wee1-specific features that maintain it in an active conformation and, together with a key substitution in its glycine-rich loop, help determine its substrate specificity.

A disulfide cross-linking strategy was used to covalently trap as a stable complex (complex N) a shortlived, kinetic intermediate in DNA polymerization. This intermediate corresponds to the product of polymerization prior to translocation. We also prepared the trapped complex that corresponds to the product of polymerization after translocation (complex P). The crosslinking method that we used is a variation of a technique developed by the Verdine and Harrison laboratories. It involves disulfide interchange between an engineered sulfhydryl group of the protein (Q258C mutation) and a disulfide-containing tether attached at the N 2 amino group of a modified dG in either the template or the primer strand of the nucleic acid. We report here a highly efficient synthesis of the precursor, bis(3-aminopropyl)disulfide dihydrochloride, used to introduce this substituent into the oligonucleotide. Efficient cross-linking takes place when the base pair containing the substituent is positioned seven registers from the dNTP-binding site (N site) and the N site is occupied. Complex N, but not complex P, is a substrate for the ATP-based excision reaction that unblocks nucleoside reverse transcriptase inhibitor (NRTI)-terminated primers and causes resistance to several NRTIs, confirming predictions that the excision reaction takes place only when the 3-end of the primer is bound at the N site. These techniques can be used for biochemical and structural studies of the mechanism of DNA polymerization, translocation, and excision-based resistance of RT to NRTIs. They may also be useful in studying other DNA or RNA polymerases or other enzymes. HIV-11 reverse transcriptase (RT) is a complex molecular machine that uses several kinetically distinct steps to incorporate a nucleotide into a growing DNA strand. It is a heterodimer composed of a larger 560-residue subunit (p66) and a smaller subunit (p51) that contains the N-terminal 440 residues of p66. Both subunits contain subdomains that were named fingers, palm, thumb, and connection, because of the similarity of p66 to a right hand. The DNA polymerase active site is located in the p66 palm subdomain and the DNA binding cleft is formed primarily by the p66 fingers, palm, and thumb subdomains. The mechanism of polymerization by RT is similar to other polymerases and involves: 1) binding of the DNA substrate to the apo-enzyme; 2) binding of dNTP and divalent metal ions (required for catalysis) to the enzyme⅐DNA complex, followed by rate-limiting conformational changes; 3) formation of a phosphodiester bond between the 3Ј-OH primer terminus and the ␣-phosphate of dNTP, followed by release of the pyrophosphate product; 4) translocation of the elongated DNA primer (for processive synthesis) from the dNTP-binding site (N site) to the priming site (P site) or release of the nucleic acid (distributive synthesis) (Fig. 1).Extensive biochemical and crystallographic studies have enhanced our understanding of the details of the mechanism of DNA polymerization. However, the translocation step rem...

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Aldo-keto reductase 1C3 (AKR1C3) catalyses the NADPH dependent reduction of carbonyl groups in a number of important steroid and prostanoid molecules. The enzyme is also over-expressed in prostate and breast cancer and its expression is correlated with the aggressiveness of the disease. The steroid products of AKR1C3 catalysis are important in proliferative signalling of hormone-responsive cells, while the prostanoid products promote prostaglandin-dependent proliferative pathways. In these ways, AKR1C3 contributes to tumour development and maintenance, and suggest that inhibition of AKR1C3 activity is an attractive target for the development of new anti-cancer therapies. Non-steroidal anti-inflammatory drugs (NSAIDs) are one well-known class of compounds that inhibits AKR1C3, yet crystal structures have only been determined for this enzyme with flufenamic acid, indomethacin, and closely related analogues bound. While the flufenamic acid and indomethacin structures have been used to design novel inhibitors, they provide only limited coverage of the NSAIDs that inhibit AKR1C3 and that may be used for the development of new AKR1C3 targeted drugs. To understand how other NSAIDs bind to AKR1C3, we have determined ten crystal structures of AKR1C3 complexes that cover three different classes of NSAID, N-phenylanthranilic acids (meclofenamic acid, mefenamic acid), arylpropionic acids (flurbiprofen, ibuprofen, naproxen), and indomethacin analogues (indomethacin, sulindac, zomepirac). The N-phenylanthranilic and arylpropionic acids bind to common sites including the enzyme catalytic centre and a constitutive active site pocket, with the arylpropionic acids probing the constitutive pocket more effectively. By contrast, indomethacin and the indomethacin analogues sulindac and zomepirac, display three distinctly different binding modes that explain their relative inhibition of the AKR1C family members. This new data from ten crystal structures greatly broadens the base of structures available for future structure-guided drug discovery efforts.