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Heteronuclear multidimensional nuclear magnetic resonance methods were used in the investigation of the structure of Delta5-3-ketosteroid isomerase homodimer complexed with 19-nortestosterone hemisuccinate in solution. Results reveal that each monomer of the enzyme consists of three alpha-helices, eight beta-strands and two beta-bulges. The three alpha-helices are situated at the concavity formed by the beta-sheet.

The influence of Asp-99 and Tyr-14 in the catalysis of ketosteroid isomerase was determined through the use of 3D 1H-13C NOESY-HSQC spectrum of Tyr-14 mutant isomerase and UV resonance Raman spectroscopy. The H-bond donation by Tyr-14 was compensated by another H-bond donor. The side chain of Asp-99 was characterized to be proximal to the aromaric ring of Tyr-14. Asp-99 donation of H-bond was stronger that that of Tyr-14, confirming the importance of Asp and Tyr residues in the formation of H-bonds on enzymes.

The tertiary structure of the Mut enzyme has been determined by distance geometry, simulated annealing and energy minimization with the program X-PLOR. The results indicate that the structure is globular and compact with the parallel portion of the beta-sheet sandwiched by two alpha-helices, forming an alpha + beta fold. A cluster of five glutamate residues form a region of electrostatic potential that constitute the metal binding site.

Nuclear magnetic resonance studies show that the binding of MutT enzyme to the nucleotides Mg2+ -AMPCPP and dGTP is anti conformation and this prevents intramolecular clashes when nucleotides with bulky substituents at the eighth position of the purine ring are hydrolyzed. The active site of MutT recognizes the nucleotides which prefer syn conformation during DNA transcription and MutT reduces the mutation rate in Escherichia coli by removing these nucleotides. It is possible that the anti conformation is essential for the MutT-catalyzed substitutions at the beta-phosphorous.

Staphylococcal nuclease (SNase) makes significant changes in its backbone arrangement to bind to nucleotide. The hydrolysis of polynucleotides occurs through a trigonal bipyramidal transition state and Arg35 and Arg87 stabilize this intermediate stage through bidentate H-bonding. Arg35 in the binary metal complex of D21E mutant of SNase binds with thymidine 3',5'-diphosphate coordination.

The Asp21 yielding Glu mutation of staphylococcal nuclease results in changed position of Ca2+ and direct coordination of Glu-43 by Ca2+, leading to loss of catalytic activity. The water ligand on Ca2+ is hydrogen bonded by Glu-43, leading to decreased dissociation and less catalytic activity. Arg-87 promotes catalysis through cooperative function of Glu-43 in orienting the attacking hydroxyl ligand.

Hfq proteins in Gram-negative bacteria play important roles in bacterial physiology and virulence, mediated by binding of the Hfq hexamer to small RNAs and/or mRNAs to post-transcriptionally regulate gene expression. However, the physiological role of Hfqs in Gram-positive bacteria is less clear. Bacillus anthracis, the causative agent of anthrax, uniquely expresses three distinct Hfq proteins, two from the chromosome (Hfq1, Hfq2) and one from its pXO1 virulence plasmid (Hfq3). The protein sequences of Hfq1 and 3 are evolutionarily distinct from those of Hfq2 and of Hfqs found in other Bacilli. Here, the quaternary structure of each B. anthracis Hfq protein, as produced heterologously in Escherichia coli, was characterized. While Hfq2 adopts the expected hexamer structure, Hfq1 does not form similarly stable hexamers in vitro. The impact on the monomer-hexamer equilibrium of varying Hfq C-terminal tail length and other sequence differences among the Hfqs was examined, and a sequence region of the Hfq proteins that was involved in hexamer formation was identified. It was found that, in addition to the distinct higher-order structures of the Hfq homologs, they give rise to different phenotypes. Hfq1 has a disruptive effect on the function of E. coli Hfq in vivo, while Hfq3 expression at high levels is toxic to E. coli but also partially complements Hfq function in E. coli. These results set the stage for future studies of the roles of these proteins in B. anthracis physiology and for the identification of sequence determinants of phenotypic complementation. [ABSTRACT FROM AUTHOR]

Coenzyme F420 is a deazaflavin hydride carrier with a lower reduction potential than most flavins. In Mycobacterium tuberculosis ( Mtb), F420 plays an important role in activating PA-824, an antituberculosis drug currently used in clinical trials. Although F420 is important to Mtb redox metabolism, little is known about the enzymes that bind F420 and the reactions that they catalyze. We have identified a novel F420-binding protein, Rv1155, which is annotated in the Mtb genome sequence as a putative flavin mononucleotide (FMN)-binding protein. Using biophysical techniques, we have demonstrated that instead of binding FMN or other flavins, Rv1155 binds coenzyme F420. The crystal structure of the complex of Rv1155 and F420 reveals one F420 molecule bound to each monomer of the Rv1155 dimer. Structural, biophysical, and bioinformatic analyses of the Rv1155-F420 complex provide clues about its role in the bacterium. [ABSTRACT FROM AUTHOR]