Bdellovibrio bacteriovorus phosphoglucose isomerase structures reveal novel rigidity in the active site of a selected subset of enzymes upon substrate binding.

Glycolysis and gluconeogenesis are central pathways of metabolism across all domains of life. A prominent enzyme in these pathways is phosphoglucose isomerase (PGI), which mediates the interconversion of glucose-6-phosphate and fructose-6-phosphate. The predatory bacterium Bdellovibrio bacteriovorus leads a complex life cycle, switching between intraperiplasmic replicative and extracellular 'hunter' attack-phase stages. Passage through this complex life cycle involves different metabolic states. Here we present the unliganded and substrate-bound structures of the B. bacteriovorus PGI, solved to 1.74 Å and 1.67 Å, respectively. These structures reveal that an induced-fit conformational change within the active site is not a prerequisite for the binding of substrates in some PGIs. Crucially, we suggest a phenylalanine residue, conserved across most PGI enzymes but substituted for glycine in B. bacteriovorus and other select organisms, is central to the induced-fit mode of substrate recognition for PGIs. This enzyme also represents the smallest conventional PGI characterized to date and probably represents the minimal requirements for a functional PGI.

The structure of Escherichia coli nitroreductase complexed with nicotinic acid: three crystal forms at 1.7 A, 1.8 A and 2.4 A resolution.

Escherichia coli nitroreductase is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Its ability to convert relatively non-toxic prodrugs such as CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide) into highly cytotoxic derivatives has led to interest in its potential for cancer gene therapy. We have determined the structure of the enzyme bound to a substrate analogue, nicotinic acid, from three crystal forms at resolutions of 1.7 A, 1.8 A and 2.4 A, representing ten non-crystallographically related monomers. The enzyme is dimeric, and has a large hydrophobic core; each half of the molecule consists of a five-stranded beta-sheet surrounded by alpha-helices. Helices F and F protrude from the core region of each monomer. There is an extensive dimer interface, and the 15 C-terminal residues extend around the opposing monomer, contributing the fifth beta-strand. The active sites lie on opposite sides of the molecule, in solvent-exposed clefts at the dimer interface. The FMN forms hydrogen bonds to one monomer and hydrophobic contacts to both; its si face is buried. The nicotinic acid stacks between the re face of the FMN and Phe124 in helix F, with only one hydrogen bond to the protein. If the nicotinamide ring of the coenzyme NAD(P)H were in the same position as that of the nicotinic acid ligand, its C4 atom would be optimally positioned for direct hydride transfer to flavin N5. Comparison of the structure with unliganded flavin reductase and NTR suggests reduced mobility of helices E and F upon ligand binding. Analysis of the structure explains the broad substrate specificity of the enzyme, and provides the basis for rational design of novel prodrugs and for site-directed mutagenesis for improved enzyme activity.

Use of an enzyme-linked immunosorbent assay to assess penetration of amoxicillin into lung secretions.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure total amoxicillin concentrations penetrating lung secretions, which were compared with "active" concentrations measured by conventional bioassay. An antibody was raised in rabbits to amoxicillin conjugated to bovine serum albumin and used in a competitive binding ELISA (sensitivity, 10 ng/ml; precision [coefficient of variation], 9%). The measurement of amoxicillin in lung secretions by using the ELISA method was verified by high-performance liquid chromatography. Amoxicillin concentrations were found to be similar in both whole sonicated sputum and sol-phase sputum obtained by ultracentrifugation following single oral doses of 3 g (4.6 mg/liter for sonicated and 4.7 mg/liter for sol-phase preparations) and 250 mg (0.23 mg/liter for both preparations). Eight patients with bronchiectasis received 500 mg of amoxicillin three times daily. On the second day of therapy (4 h after the morning dose), the mean concentration of amoxicillin in sputum was 0.88 mg/liter (standard error of the mean [SEM], 0.11) by ELISA and 0.40 mg/liter (SEM, 0.05) by bioassay, suggesting a significant degree of local inactivation. This difference between total and active amoxicillin levels was found to correlate significantly (r = 0.693; P less than 0.05) with beta-lactamase levels (mean, 29.5 mU/ml; SEM, 9.4). A pharmacokinetic study on day 3 revealed maximum levels in secretions 2 to 4 h after dosing (mean, 1.36 mg/liter; SEM, 0.26). At the end of successful therapy (day 14), total and active levels were lower (mean, 0.48 mg/liter; SEM, 0.11 [total]; mean, 0.21 mg/liter; SEM, 0.06 [active]); this result was associated with a reduction in lung inflammation (decreased serum-derived albumin in the lung secretions). In conclusion, antibiotic penetration is partly dependent on the degree of lung inflammation. The differences observed in total and active levels of amoxicillin and the relationship to beta-lactamase activity in sputum suggest why higher doses of antibiotic may be required to produce a therapeutic response in some patients.