Three-dimensional structure of lipoamide dehydrogenase from Pseudomonas fluorescens at 2.8 A resolution. Analysis of redox and thermostability properties

The structure of Pseudomonas fluorescens lipoamide dehydrogenase, a dimeric flavoenzyme with a molecular mass of 106,000 daltons, was solved by the molecular replacement method and refined to an R-factor of 19.4% at 2.8 A resolution. The root-mean-square difference from ideal values for bonds and angles is 0.019 A and 3.8 degrees, respectively. The structure is closely related to that of the same flavoprotein from Azotobacter vinelandii. The root-mean-square difference for 932 C alpha atoms is 0.64 A, with 84% sequence identity. The residues in the active site are identical, while 89% of the interface residues are the same in the two enzymes. A few structural variations provide the basis for the differences in thermostability and redox properties between the two homologous proteins. Particularly, in the A. vinelandii molecule a threonine to alanine (T452A) mutation leaves a buried carbonyl oxygen, located at the subunit interface and in proximity of the flavin ring, unpaired to any H-bond donor, probably providing an explanation for the lower stability of the A. vinelandii enzyme with respect to the P. fluorescens enzyme. Six surface loops, which previously could not be accurately positioned in the A. vinelandii structure, are well defined in P. fluorescens lipoamide dehydrogenase. On the basis of the P. fluorescens structure, the six loops could be correctly defined also in the A. vinelandii enzyme. This is an unusual case where similar refinement methodologies applied to two crystal forms of closely related proteins led to electron density maps of substantially different quality. The correct definition of these surface residues is likely to be an essential step for revealing the structural basis of the interactions between lipoamide dehydrogenase and the other members of the pyruvate dehydrogenase multienzyme complex.     

Treatment of cutout of a lag screw of a dynamic hip screw in an intertrochanteric fracture

Sixteen consecutive patients with cutout of a lag screw of a dynamic hip screw fixation in an intertrochanteric fracture were treated with reinsertion of a lag screw, bone cement supplementation in the neck-trochanter, and subtrochanteric valgus osteotomy. Postoperatively, patients were permitted to ambulate with protected weight-bearing. Fourteen patients were followed-up for at least 1 year (median 2 years; range 1-3 years), and all had a solid union. The union period took a median of 5 months, with a range of 3-7 months. Usually, union of an intertrochanteric fracture was faster than that of subtrochanteric osteotomy (P < 0.01). There were no complications of wound infection, loss of reduction, cutout of a lag screw, or osteonecrosis of the femoral head. From clinical and theoretical considerations, we conclude that despite cutout of a lag screw of a dynamic hip screw fixation being difficult to treat, out technique still can provide an excellent outcome. Therefore, we strongly recommend its wide use.     

The alcohol deprivation effect in the alcohol-preferring P rat under free-drinking and operant access conditions

The alcohol-deprivation effect (ADE) was examined under 4-hr operant and 24-hr free-choice alcohol access in the alcohol-preferring (P) rat after deprivation intervals from 2 to 4 weeks. Results indicated that adult male P rats responding for 6 weeks on a concurrent FR-5/FR-1 schedule of reinforcement for alcohol and water, respectively, and then deprived of alcohol for 2 weeks, demonstrated a 40% increase in alcohol responding during the first 60 min of alcohol reinstatement. The alcohol deprivation effect was temporary, however, as responding did not differ from baseline levels on the second day of reinstatement. In a second experiment, weanling male and female P rats received 7 weeks of continuous access to alcohol, beginning at 21 days of age, and were then deprived of alcohol for 4 weeks. On the first day of alcohol reinstatement, P rats exhibited a 40% to 45% increase from baseline alcohol drinking levels, with alcohol intake returning to baseline levels by the 3rd day of reinstatement. Although alcohol intake was higher in females than in males when adjustment was made for body weight, there were no gender differences in the magnitude of the alcohol deprivation effect. Taken together, these results indicate that the ADE is a long-lasting phenomenon that occurs under both operant and continuous access conditions in the P rat, and thus these rats may be useful models for the study of factors involved in relapse of alcohol drinking.     

Stereochemical course of enzymatic enolpyruvyl transfer and catalytic conformation of the active site revealed by the crystal structure of the fluorinated analogue of the reaction tetrahedral intermediate bound to the active site of the C115A mutant of MurA

MurA (UDP-GlcNAc enolpyruvyl transferase), the first enzyme in bacterial peptidoglycan biosynthesis, catalyzes the enolpyruvyl transfer from phosphoenolpyruvate (PEP) to the 3'-OH of UDP-GlcNAc by an addition-elimination mechanism that proceeds through a tetrahedral ketal intermediate. The crystal structure of the Cys115-to-Ala (C115A) mutant of Escherichia coli MurA complexed with a fluoro analogue of the tetrahedral intermediate revealed the absolute configuration of the adduct and the stereochemical course of the reaction. The fluorinated adduct was generated in a preincubation of wild-type MurA with (Z)-3-fluorophosphoenolpyruvate (FPEP) and UDP-GlcNAc and purified after enzyme denaturation. The fluorine substituent stabilizes the tetrahedral intermediate toward decomposition by a factor of 10(4)-10(6), facilitating manipulation of the adduct. The C115A mutant of MurA was utilized to avoid the microheterogeneity that arises in the wild-type MurA from the attack of Cys115 on C-2 of FPEP in competition with the formation of the fluorinated adduct. The crystal structure of the complex was determined to 2.8 A resolution, and the absolute configuration at C-2 of the adduct was found to be 2R. Thus, addition of the 3'-OH of UDP-GlcNAc is to the 2-si face of FPEP, corresponding to the 2-re face of PEP. Given the previous observation that, in D2O, the addition of D+ to C-3 of PEP proceeds from the 2-si face [Kim, D. H., Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380-6381], the addition across the double bond of PEP is anti. Also, because the overall stereochemical course has been shown to be either anti/syn or syn/anti [Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 7329-7337], it now follows that the stereochemistry of elimination of H+ from C-3 and Pi from C-2 of the tetrahedral intermediate of the reaction is syn.     

T lymphocyte responses to anti-neutrophil cytoplasmic autoantibody (ANCA) antigens are present in patients with ANCA-associated systemic vasculitis and persist during disease remission

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel has been identified in the cardiac muscle of a number of mammalian species, including humans. The goal of this study was to begin quantifying the structural requirements necessary for arylaminobenzoate block of the CFTR channel. The cardiac cAMP-dependent Cl- current (ICl) was measured using the whole-cell arrangement of the patch-clamp technique in guinea pig ventricular myocytes during stimulation of protein kinase A with forskolin. At drug concentrations below the IC50 value for channel block, reduction of ICl by the arylaminobenzoates occurred in a strongly voltage-dependent manner with preferential inhibition of the inward currents. At higher drug concentrations, block of both the inward and outward ICl was observed. Increasing the length of the carbon chain between the benzoate and phenyl rings of the arylaminobenzoates resulted in a marked increase in drug block of the channel, with IC50 values of 47, 17, and 4 microM for 2-benzylamino-5-nitro-benzoic acid, 5-nitro-2-(2-phenylethylamino)-benzoic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), respectively. Increasing the carbon chain length further with the compound 5-nitro-2-(4-phenylbutylamino)-benzoic acid, caused no additional increase in the potency of drug block (IC50 = 4 microM). Inhibition of ICl by the arylaminobenzoates was modulated by the pH of the external solution; increasing the pH from 7.4 to 10.0 greatly weakened NPPB block, whereas decreasing the pH to 6.4 enhanced block. In addition, block of ICl was observed during intracellular dialysis of NPPB, and this action was not affected by raising the external pH.     

Concerted evolution in an egg receptor for a rapidly evolving abalone sperm protein

Gamete interactions during fertilization exhibit species specificity. In abalone, the sperm protein lysin species-specifically creates a hole in the egg envelope. Lysin evolves rapidly by positive Darwinian selection. Evolution of the egg receptor for lysin provides the selective pressure for lysin's divergence. The egg receptor for lysin is a tandemly repeated sequence that evolves by concerted evolution. Concerted evolution in the egg receptor could explain the rapid, adaptive evolution in sperm lysin and may provide an underlying molecular mechanism that gives rise to species-specific fertilization.     

Analysis of fluoromethyl group chirality establishes a common stereochemical course for the enolpyruvyl transfers catalyzed by EPSP synthase and UDP-GlcNAc enolpyruvyl transferase

The stereochemistry of transient methyl group formation at C-3 of phosphoenolpyruvate (PEP) in the reaction catalyzed by 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase has been examined using the pseudosubstrates, (E)- and (Z)-3-fluorophosphoenolpyruvate (FPEP). Kinetically stable, chiral [1H, 2H]fluoromethyl analogs of the reaction tetrahedral intermediate were isolated and subjected to decomposition and stereochemical analysis. EPSP synthase was found to catalyze the 2-re face addition of solvent-derived hydrogen to C-3 of FPEP (corresponding to the 2-si face of PEP). Comparison of these data with prior analogous work on the MurA reaction [Kim, D.H., Lees, W.J., & Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380-6381] suggests that the two enolpyruvyl transferases share a common stereochemical course, further strengthening the mechanistic, structural, and evolutionary relationship between the two enzymes.