The influence of His94 and Pro149 in modulating the activity of V. cholerae DsbA.

DsbA is the primary catalyst of disulfide bond formation in the periplasm of gram-negative bacteria. Numerous theoretical and experimental studies have been undertaken to determine the molecular mechanisms by which DsbA acts as a potent oxidant, whereas the homologous cytoplasmic protein, thioredoxin, acts as a reductant. Many of these studies have focused on the nature of the two residues that lie between the active-site cysteines. Although these are clearly important, they are not solely responsible for the differences in activity between these thiol-disulfide oxidoreductases. Q97 in the helical domain of E. coli DsbA has been implicated in influencing the redox potential of E. coli DsbA. In V. cholerae DsbA, the analogous residue is H94. In this study, the effect of H94 on the oxidase activity of DsbA is examined, along with the role of the conserved cis-proline residue P149. The DsbA mutant H94L shows a nearly fourfold increase in activity over the wild-type enzyme. To our knowledge, this is the first time an increase in the normal activity of a thiol-disulfide oxidoreductase has been reported. Potential reasons for this increase in activity are discussed.

In vivo reaction of affinity-tag-labelled epidermin precursor peptide with flavoenzyme EpiD.

The Staphylococcus epidermidis genes encoding the His-tag-labelled epidermin precursor peptide EpiA and the flavoenzyme EpiD or the mutant protein EpiD-G93D, which lacks the coenzyme, were co-expressed and the proteins were synthesized in vivo in Escherichia coli. Only in the presence of EpiD was the precursor peptide converted to a reaction product with a decrease in mass of 44-46 Da. This result confirms the in vitro experiments carried out with purified EpiA and purified EpiD from Staphylococcus epidermidis [Kupke et al. (1994) J. Biol. Chem. 269, 5653-5659]. EpiD catalyzes the oxidative decarboxylation of the C-terminal cysteine residue of EpiA to a [Z]-enethiol structure. In the presence of EpiD, the amount of purified (modified) peptide EpiA was several-fold higher than in the presence of EpiD-G93D, indicating that the stabilization of EpiA against proteolysis is due to an interaction with EpiD or to the presence of the C-terminal modification. The presented experimental approach will be valuable for the analysis of enzymes that catalyze posttranslational modification reaction of peptides and proteins.

Purification and characterization of EpiA, the peptide substrate for post-translational modifications involved in epidermin biosynthesis.

For the investigation of enzymes involved in epidermin biosynthesis it is necessary to produce sufficient amounts of preepidermin (EpiA) as a substrate and to design EpiA detection systems. Therefore, EpiA was expressed in Escherichia coli using a malE-epiA fusion. The identity of purified EpiA was confirmed by ion spray mass spectrometry and amino acid sequencing. For EpiA detection, anti-EpiA antisera were raised. Upon prolonged incubation, factor Xa not only cleaved EpiA from the fusion protein, but also less efficiently cleaved EpiA internally between R-1 and I+1. The internal factor Xa cleavage site of EpiA was masked by altering the sequence -A(-4)-E-P-R(-1)- to -A(-4)-E-P-Q(-1)- by site-directed mutagenesis.

Differential sensitivity of the WAIS and a modified Halstead-Reitan battery to severity of brain dysfunction in epilepsy.

The present study examined the diagnostic sensitivity of the WAIS relative to a modified Halstead-Reitan test battery by comparing the accuracy of these instruments in differentiating pseudoneurologic control subjects from two groups of epileptics which were believed to differ in degree of underlying brain pathology. A total of 96 subjects were equally represented in the control, mildly impaired, and moderately impaired study groups. Separate three-group and two-group discriminant function analyses were performed for the two sets of measures. Comparisons of kappa coefficients indicated that the WAIS was inferior in classification accuracy to the neuropsychological tests in making the three-group discriminations and in separating the mildly impaired subjects from the moderately impaired group. These findings suggest that the WAIS may be inferior in discriminatory power relative to a wider array of neuropsychological measures than has heretofore been evaluated. Furthermore, these data imply that the degree of underlying brain involvement associated with a seizure disorder may be determined best from neuropsychological tests rather than from a general measure of intellectual ability, such as the WAIS.

Relative influence of subject variables and neurological parameters on neuropsychological performance of adult seizure patients.

We present the results of a study examining relationships between subject characteristics (age, education, sex, race) and neurological factors on neuropsychological test performance of adult seizure patients. Test scores from a modified and expanded Halstead-Reitan test battery for 250 epileptics served as criterion variables for sets of multiple regression analyses using subject characteristics and neurological parameters as predictors. Initially, subjects were divided into two subsamples and separate analyses for subject and neurological variables were pursued. Double cross-validated results indicated that test performance was significantly related to both sets of predictor variables, yet the magnitudes of relationship were rather consistently highest for demographic attributes. The relative contribution of individual predictor variables subsequently was explored in a second set of multiple regression analyses examining both subject and neurological variables within the combined patient sample. Results indicated that one or more subject characteristic plus one or more neurological variable accounted for a significant amount of test variance for almost all of the measures under consideration. A subject variable was entered first in step-wise regression analyses for 78 % of the measures, with education proving to be the most potent predictor of test status. In general, these data support the need for test norms which make adjustments for nonneurological subject attributes.

On being informed you are HIV positive: experiences of Navy service members.

This study addressed the experience of being told that one has become infected with the human immunodeficiency virus (HIV) while serving in the United States Navy. Responses to a questionnaire, administered to 150 HIV-positive service members, indicated that feelings of fear, shock, disbelief, and embarrassment were experienced by study participants upon learning of their HIV-positive status. The manner in which their HIV diagnosis was disclosed was generally viewed in favorable terms and more so in recent years relative to the earliest days of the Navy's HIV program. Having a medical officer as a disclosing official was associated with more negative experiences than was the case for other categories of disclosing officials. Lastly, post-disclosure events were often excessively stressful, and no improvement in this regard over 6 years of the Navy's HIV program was evident.

Molecular characterization of the 4'-phosphopantothenoylcysteine decarboxylase domain of bacterial Dfp flavoproteins.

The NH(2)-terminal domain of the bacterial flavoprotein Dfp catalyzes the decarboxylation of (R)-4'-phospho-N-pantothenoylcysteine to 4'-phosphopantetheine, a key step in coenzyme A biosynthesis. Dfp proteins, LanD proteins (for example EpiD, which is involved in epidermin biosynthesis), and the salt tolerance protein AtHAL3a from Arabidopsis thaliana are homooligomeric flavin-containing Cys decarboxylases (HFCD protein family). The crystal structure of the peptidyl-cysteine decarboxylase EpiD complexed with a pentapeptide substrate has recently been determined. The peptide is bound by an NH(2)-terminal substrate binding helix, residue Asn(117), which contacts the cysteine residue of the substrate, and a COOH-terminal substrate recognition clamp. The conserved motif G-G/S-I-A-X-Y-K of the Dfp proteins aligns partly with the substrate binding helix of EpiD. Point mutations within this motif resulted in loss of coenzyme binding (G14S) or in significant decrease of Dfp activity (G15A, I16L, A17D, K20N, K20Q). Exchange of Asn(125) of Dfp, which corresponds to Asn(117) of EpiD, and exchange of Cys(158), which is within the proposed substrate recognition clamp of Dfp, led to inactivity of the enzyme. Molecular analysis of the conditional lethality of the Escherichia coli dfp-707 mutant revealed that the single point mutation G11D of Dfp is related to decreased amounts of soluble Dfp protein at 37 degrees C.

Post-translational modifications of lantibiotics.

Several newly reported post-translational modification reactions are involved in lantibiotic biosynthesis. A short overview of the present knowledge on the post-translational modifications and on the enzymes involved in lantibiotic biosynthesis is given. The oxidative decarboxylation of the epidermin precursor peptide EpiA is described in detail. The FMN-containing oxidoreductase EpiD is involved in the formation of the C-terminal S-[(Z)-2-aminovinyl]-D-cysteine residue of epidermin: under reducing conditions the side chain of the C-terminal cysteine residue of EpiA is converted to an enethiol. EpiD has no absolute substrate specificity and can be used for modification of peptides having the C-terminal consensus motif [V/I/L/(M)/F/Y/W]-[A/S/V/T/C/(I/L)]-C.

The enethiolate anion reaction products of EpiD. Pka value of the enethiol side chain is lower than that of the thiol side chain of peptides.

One of the steps involved in the biosynthesis of the lantibiotic epidermin is the oxidative decarboxylation reaction of peptides catalyzed by the flavoenzyme EpiD. EpiD catalyzes the formation of a (Z)-enethiol derivative from the C-terminal cysteine residue of the precursor peptide of epidermin and related peptides. The UV-visible spectra of the reaction products of EpiD are pH-dependent, indicating that the enethiol side chain is converted to an enethiolate anion. The pKa value of the enethiol group was determined to be 6.0 and is substantially lower than the pKa value of the thiol side chain of cysteine residues. The increased acid strength of the enethiol side chain compared with that of the thiol group is attributed to the resonance stabilization of the negative charge of the anion.

Expression, purification, and characterization of EpiC, an enzyme involved in the biosynthesis of the lantibiotic epidermin, and sequence analysis of Staphylococcus epidermidis epiC mutants.

The plasmid-encoded epidermin biosynthetic gene epiC of Staphylococcus epidermidis Tü3298 was expressed in Escherichia coli by using the T7 RNA polymerase-promoter system, and the gene product EpiC was identified by Western blotting (immunoblotting) with an anti-EpiC-peptide antiserum. EpiC was a hydrophobic but soluble protein. EpiC was purified by hydrophobic-interaction chromatography. The determined amino-terminal amino acid sequence was M I N I N N I .... The electrophoretic migration behavior of EpiC depended on the oxidation state of the enzyme, indicating the formation of an intramolecular disulfide bridge between C-274 and C-321. The cysteine residues in the motifs WC-274YG and C-321HG of EpiC are conserved in all lantibiotic enzymes of the C type (so-called LanC proteins) and in the CylM protein. Mutated epiC genes from S. epidermidis epiC mutants were cloned and expressed in E. coli. Sequence analysis revealed that the mutations occurred in the two motifs -S-X-X-X-G-X-X-G- and -N-X-G-X-A-H-G-X-X-G-, which are conserved in all LanC proteins. For the investigation of EpiC-EpiA interactions, precursor peptide EpiA was coupled to N-hydroxysuccinimide-activated Sepharose High Performance Material (HiTrap). Under reducing conditions, EpiC was retarded on the EpiA-HiTrap column. In the incubation experiments, EpiC did not react with EpiA, with proepidermin, or with oxidative decarboxylated peptides.

WAIS and neuropsychological tests: common and unique variance within an epileptic population.

Neuropsychological assessment batteries, which traditionally require lengthy administration times, may be composed of multiple tests which measure essentially the same performance attributes. Test redundancies of this nature compromise the adequacy of a test battery from a cost containment perspective. The present study evaluated the common and unique variance of the WAIS relative to a modified and expanded Halstead-Reitan test battery to determine if information obtained from the WAIS is sufficiently nonredundant to justify its inclusion as part of this assessment battery. Cross-validated estimates of bivariate and multivariate relationships between these instruments were generated from two samples (n = 125) of epileptics. Results indicated statistically significant redundancies, yet, when interpreted from a clinical perspective it was evident that neuropsychological tests could not accurately duplicate information provided by the WAIS. Accordingly, it was not recommended that the WAIS be eliminated from this test battery in order to reduce test administration time.

Serine protease EpiP from Staphylococcus epidermidis catalyzes the processing of the epidermin precursor peptide.

The function of serine protease EpiP in epidermin biosynthesis was investigated. Epidermin is synthesized as a 52-amino-acid precursor peptide, EpiA, which is posttranslationally modified and processed to the mature 22-amino-acid peptide antibiotic. epiP was expressed in Staphylococcus carnosus with xylose-regulated expression vector pCX15. The cleavage of the unmodified EpiA precursor peptide to leader peptide and proepidermin by EpiP-containing culture filtrates of S. carnosus (pCX15epiP) was followed by reversed-phase chromatography and subsequent electrospray mass spectrometry.

Neuropsychological impairment and behavioral limitations exhibited within an alcohol treatment program.

The present study investigated the generalizability of neuropsychological test data to behavioral problems/limitations exhibited by alcoholics within an inpatient treatment program. Ratings supplied by alcohol counselors of problematic behaviors were used to form two groups (N = 40) representing behaviorally impaired (B1) and behaviorally unimpaired (BU) alcoholics. The neuropsychological performance of BI subjects was found to be impaired, relative to BU subjects, on composite measures of motor skill, problem solving, psychomotor speed, and memory. BI subjects, relative to the BU group, were also found to have significantly longer histories of alcoholism, more frequent neurological examination abnormalities, and higher incidences of suboptimal nutrition. Taken as a whole, these data suggest that neuropsychological measures may have some potential for assisting in the generating of valid inferences regarding both underlying cerebral pathology and the behavioral consequences of such as expressed within the alcohol treatment milieu.

Crystal structure of the peptidyl-cysteine decarboxylase EpiD complexed with a pentapeptide substrate.

Epidermin from Staphylococcus epidermidis Tü3298 is an antimicrobial peptide of the lantibiotic family that contains, amongst other unusual amino acids, S:-[(Z:)- 2-aminovinyl]-D-cysteine. This residue is introduced by post-translational modification of the ribosomally synthesized precursor EpiA. Modification starts with the oxidative decarboxylation of its C-terminal cysteine by the flavoprotein EpiD generating a reactive (Z:)-enethiol intermediate. We have determined the crystal structures of EpiD and EpiD H67N in complex with the substrate pentapeptide DSYTC at 2.5 A resolution. Rossmann-type monomers build up a dodecamer of 23 point symmetry with trimers disposed at the vertices of a tetrahedron. Oligomer formation is essential for binding of flavin mononucleotide and substrate, which is buried by an otherwise disordered substrate recognition clamp. A pocket for the tyrosine residue of the substrate peptide is formed by an induced fit mechanism. The substrate contacts flavin mononucleotide only via Cys-Sgamma, suggesting its oxidation as the initial step. A thioaldehyde intermediate could undergo spontaneous decarboxylation. The unusual substrate recognition mode and the type of chemical reaction performed provide insight into a novel family of flavoproteins.

Improved purification and biochemical properties of phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis.

Monophosphatidylinositol inositol phosphohydrolase (phosphatidylinositol-specific phospholipase C. PtdIns-PLC. EC 3.1.4.10) has been purified from a Bacillus thuringiensis culture supernatant and from the cellular fraction of a recombinant Escherichia coli clone containing the PtdIns-PLC gene from B. thuringiensis. The two-step purification procedure involved ion-exchange chromatography on DEAE-Sepharose followed by separation on a Mono-Q/FPLC-column with yields of 32% and 50%, respectively. The molecular mass was determined to be 34 kDa by SDS/PAGE. The isoelectric point of the enzyme was 5.15. The amino-terminal sequences were shown to be identical for the enzymes purified from both organisms. PtdIns-PLC was inhibited by divalent cations using mixed micelles of Triton X-100 and pure phosphatidylinositol. PtdIns-PLC activity was detectable on polyacrylamide gels by activity staining on phosphatidylinostiol-containing agarose.

Molecular characterization and sequence of phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis.

The gene encoding monophosphatidylinositol inositol phosphohydrolase (PI-specific phospholipase C, PI-PLC) of Bacillus thuringiensis was cloned in Staphylococcus carnosus TM300. The complete coding region comprises 987 base pairs corresponding to a precursor protein of 329 amino acids (molecular weight, 38,095). The NH2-terminal sequence of the purified enzyme from Escherichia coli indicated that the mature PI-PLC consists of 299 amino acid residues with a molecular weight of 34,586. Polyacrylamide gel electrophoresis revealed the same molecular weight for the purified enzyme isolated from the DNA-donor strain of B. thuringiensis and from the E. coli clone. By computer analysis, the secondary structure was predicted. The enzyme from the E. coli recombinant shows no activity on other phospholipids and sphingomyelin. The cleaving specificity of PI-PLC was examined by thin layer chromatography.