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.

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.

Arabidopsis thaliana flavoprotein AtHAL3a catalyzes the decarboxylation of 4'-Phosphopantothenoylcysteine to 4'-phosphopantetheine, a key step in coenzyme A biosynthesis.

The Arabidopsis thaliana flavoprotein AtHAL3a is related to plant growth and salt and osmotic tolerance. AtHAL3a shows sequence homology to the bacterial flavoproteins EpiD and Dfp. EpiD, Dfp, and AtHAL3a are members of the homo-oligomeric flavin-containing Cys decarboxylase (HFCD) protein family. We demonstrate that AtHAL3a catalyzes the decarboxylation of (R)-4'-phospho-N-pantothenoylcysteine to 4'-phosphopantetheine. This key step in coenzyme A biosynthesis is catalyzed in bacteria by the Dfp proteins. Exchange of His-90 of AtHAL3a for Asn led to complete inactivation of the enzyme. Dfp and AtHAL3a are characterized by a shortened substrate binding clamp compared with EpiD. Exchange of the cysteine residue of the conserved ACGD motif of this binding clamp resulted in loss of (R)-4'-phospho-N-pantothenoylcysteine decarboxylase activity. Based on the crystal structures of EpiD H67N with bound substrate peptide and of AtHAL3a, we present a model for the binding of (R)-4'-phospho-N-pantothenoylcysteine to AtHAL3a.

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.

Molecular characterization of lantibiotic-synthesizing enzyme EpiD reveals a function for bacterial Dfp proteins in coenzyme A biosynthesis.

The lantibiotic-synthesizing flavoprotein EpiD catalyzes the oxidative decarboxylation of peptidylcysteines to peptidyl-aminoenethiols. The sequence motif responsible for flavin coenzyme binding and enzyme activity is conserved in different proteins from all kingdoms of life. Dfp proteins of eubacteria and archaebacteria and salt tolerance proteins of yeasts and plants belong to this new family of flavoproteins. The enzymatic function of all these proteins was not known, but our experiments suggested that they catalyze a similar reaction like EpiD and/or may have similar substrates and are homododecameric flavoproteins. We demonstrate that the N-terminal domain of the Escherichia coli Dfp protein catalyzes the decarboxylation of (R)-4'-phospho-N-pantothenoylcysteine to 4'-phosphopantetheine. This reaction is essential for coenzyme A biosynthesis.

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.

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.

The biosynthesis of the lantibiotics epidermin, gallidermin, Pep5 and epilancin K7.

Lantibiotics are antibiotic peptides that contain the rare thioether amino acids lanthionine and/or methyllanthionine. Epidermin, Pep5 and epilancin K7 are produced by Staphylococcus epidermidis whereas gallidermin (6L-epidermin) was isolated from the closely related species Staphylococcus gallinarum. The biosynthesis of all four lantibiotics proceeds from structural genes which code for prepeptides that are enzymatically modified to give the mature peptides. The genes involved in biosynthesis, processing, export etc. are found in gene clusters adjacent to the structural genes and code for transporters, immunity functions, regulatory proteins and the modification enzymes LanB, LanC and LanD, which catalyze the biosynthesis of the rare amino acids. LanB and LanC are responsible for the dehydration of the serine and threonine residues to give dehydroalanine and dehydrobutyrine and subsequent addition of cysteine SH-groups to the dehydro amino acids which results in the thioether rings. EpiD, the only LanD enzyme known so far, catalyzes the oxidative decarboxylation of the C-terminal cysteine of epidermin which gives the C-terminal S-aminovinylcysteine after addition of a dehydroalanine residue.

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.

Protein engineering of lantibiotics.

Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases be a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply. This paper describes the development of expression systems for the structural lantibiotic genes for nisin A, nisin Z, gallidermin, epidermin and Pep5, and gives examples of recently produced site-directed mutants of these lantibiotics. Characterization of the mutants yielded valuable information on biosynthetic requirements for production. Moreover, regions in the lantibiotics were identified that are of crucial importance for antimicrobial activity. Eventually, this knowledge will lead to the rational design of lantibiotics optimally suited for fighting specific undesirable microorganisms. The mutants are of additional value for studies directed towards the elucidation of the mode of action of lantibiotics.

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.

Isolation and characterization of genetically engineered gallidermin and epidermin analogs.

Gallidermin (Gdm) and epidermin (Epi) are highly homologous tetracyclic polypeptide antibiotics that are ribosomally synthesized by a Staphylococcus gallinarum strain and a Staphylococcus epidermidis strain, respectively. These antibiotics are secreted into media and are distinguished by the presence of the unusual amino acids lanthionine, 3-methyllanthionine, didehydrobutyrine, and S-(2-aminovinyl)-D-cysteine, which are formed by posttranslational modification. To study the substrate specificities of the modifying enzymes and to obtain variants that exhibit altered or new biological activities, we changed certain amino acids by performing site-specific mutagenesis with the Gdm and Epi structural genes (gdmA and epiA, respectively). S. epidermidis Tü3298/EMS6, an epiA mutant of the Epi-producing strain, was used as the expression host. This mutant synthesized Epi, Gdm, or analogs of these antibiotics when the appropriate genes were introduced on a plasmid. No Epi or Gdm analogs were isolated from the supernatant when (i) hydroxyamino acids involved in thioether amino acid formation were replaced by nonhydroxyamino acids (S3N and S19A); (ii) C residues involved in thioether bridging were deleted (delta C21, C22 and delta C22); or (iii) a ring amino acid was replaced by an amino acid having a completely different character (G10E and Y20G). A strong decrease in production was observed when S residues involved in thioether amino acid formation were replaced by T residues (S16T and S19T). A number of conservative changes at positions 6, 12, and 14 on the Gdm backbone were tolerated and led to analogs that had altered biological properties, such as enhanced antimicrobial activity (L6V) or a remarkable resistance to proteolytic degradation (A12L and Dhb14P). The T14S substitution led to simultaneous production of two Gdm species formed by incomplete posttranslational modification (dehydration) of the S-14 residue. The fully modified Dhb14Dha analog exhibited antimicrobial activity similar to that of Gdm, whereas the Dhb14S analog was less active. Both peptides were more sensitive to tryptic cleavage than Gdm was.

Oxidative decarboxylation of peptides catalyzed by flavoprotein EpiD. Determination of substrate specificity using peptide libraries and neutral loss mass spectrometry.

The flavoprotein EpiD catalyzes the COOH-terminal oxidative decarboxylation of the lantibiotic precursor peptide EpiA. Variations of the COOH-terminal heptapeptide S1FNSYCC7 of EpiA were used for determining the substrate specificity of EpiD. When Cys7 was replaced by serine, cysteine-amide, homocysteine, or a thioether amino acid residue, no reaction with EpiD was observed. Heptapeptide libraries with one variable amino acid residue at positions 1-7 of the peptide substrate S1FNSYCC7 were incubated with EpiD, and the reaction products were identified by neutral loss mass spectrometry. When the penultimate cysteine residue Cys6 of the substrate peptide was replaced with Ser, Thr, Ala, or Val, the reaction still occurred. Tyr5 could be replaced with other hydrophobic amino acid residues. Mass spectrometry was used to compare the kinetics of the reaction of EpiD with various peptides. Peptide sequencing of the reaction products was performed by tandem mass spectrometry, confirming that the last cysteine residue was modified. The removal of the acid COOH-terminal carboxyl group was confirmed by determination of the isoelectric points of the reaction products. To study the interaction between EpiA and EpiD, EpiA was coupled to N-hydroxysuccinimide-activated Sepharose HiTrap material; EpiD was only retarded under reducing conditions.

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.

Producer immunity towards the lantibiotic Pep5: identification of the immunity gene pepI and localization and functional analysis of its gene product.

The lantibiotic Pep5 is produced by Staphylococcus epidermidis 5. Pep5 production and producer immunity are associated with the 20-kb plasmid pED503. A 1.3-kb KpnI fragment of pED503, containing the Pep5 structural gene pepA, was subcloned into the Escherichia coli-Staphylococcus shuttle vector pCU1, and the recombinant plasmid pMR2 was transferred to the Pep5- and immunity-negative mutant S. epidermidis 5 Pep5- (devoid of pED503). This clone did not produce active Pep5 but showed the same degree of insensitivity towards Pep5 as did the wild-type strain. Sequencing of the 1.3-kb KpnI-fragment and analysis of mutants demonstrated the involvement of two genes in Pep5 immunity, the structural gene pepA itself and pepI, a short open reading frame upstream of pepA. To identify the 69-amino-acid pepI gene product, we constructed an E. coli maltose-binding protein-PepI fusion clone. The immunity peptide PepI was detected in the soluble and membrane fractions of the wild-type strain and the immune mutants (harboring the plasmids pMR2 and pMR11) by immunoblotting with anti-maltose-binding protein-PepI antiserum. Strains harboring either pepI without pepA or pepI with incomplete pepA were not immune and did not produce PepI. Washing the membrane with salts and EDTA reduced the amount of PepI in this fraction, and treatment with Triton X-100 almost completely removed the peptide. Furthermore, PepI was hydrolyzed by proteases added to osmotically stabilized protoplasts. This suggests that PepI is loosely attached to the outside of the cytoplasmic membrane. Proline uptake and efflux experiments with immune and nonimmune strains also indicated that PepI may act at the membrane site.

Mass spectroscopic analysis of a novel enzymatic reaction. Oxidative decarboxylation of the lantibiotic precursor peptide EpiA catalyzed by the flavoprotein EpiD.

The epidermin biosynthetic reaction between the flavoprotein EpiD and the precursor peptide EpiA was investigated by reversed-phase chromatography and ion spray mass spectrometry. Several products with molecular masses 46 and 104 Da less than that of EpiA were observed; these results were confirmed by using an MBP-EpiD fusion protein as enzyme and the mutant peptides EpiAR-1Q and K-EpiA as substrates. The reaction was inhibited by Zn2+ ions. Modifications were localized in the C-terminal fragment of EpiA as shown by factor Xa cleavage of the products followed by mass spectrometry analysis. In addition, EpiD reacted with the precursor peptides and with proepidermin, indicating that the leader peptide is not necessary for the recognition of EpiA by EpiD. Sequence analysis of modified proepidermin revealed that at least the amino acids Ile(+1)-Tyr+20 are unmodified. The observed decrease in mass of 46 Da and the modification at the C terminus of EpiA is in agreement with the proposed enzymatic function of EpiD, the oxidative decarboxylation of the precursor peptide. In addition, the increased absorbance at 260 nm of the modified peptides indicates the presence of a thioenol group in the C-terminal proepidermin.

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.

Regulation of epidermin biosynthetic genes by EpiQ.

We investigated the role of epiQ in the biosynthesis of the lantibiotic epidermin. epiQ was essential for epidermin production. It was shown that EpiQ controls epidermin production by transcriptionally activating the epiA promoter, used for transcription of most of the epidermin biosynthetic genes. Additional copies of epiQ increased epidermin production in the epidermin-producing wild-type strain Staphylococcus epidermidis Tü3298. The epiA promoter region was characterized by primer extension analysis. Two inverted repeats, putative operator sites for EpiQ binding, are located upstream of the -35 region and one is localized downstream of the -10 region. Crude protein extracts from S. epidermidis Tü3298 and epiQ expressing Escherichia coli cells led to gel mobility shifts of a DNA fragment bearing the inverted repeat which is located immediately upstream of the -35 region. DNA fragments bearing the other two inverted repeats were not shifted. The epiQ gene product could be detected by overexpression in the E. coli T7 system using antiserum raised against synthetic peptides of EpiQ. Furthermore, EpiQ, like other DNA-binding proteins, was shown to bind strongly to heparin sepharose.

Purification and characterization of EpiD, a flavoprotein involved in the biosynthesis of the lantibiotic epidermin.

The plasmid-encoded epidermin biosynthesis gene, epiD, of Staphylococcus epidermidis Tü3298 was expressed in Escherichia coli by using both the malE fusion system and the T7 RNA polymerase-promoter system. EpiD was identified by Western blotting (immunoblotting) with anti-maltose-binding protein (MBP)-EpiD antiserum. EpiD and the MBP-EpiD fusion protein, which were mainly present in the soluble protein fraction, were purified from the respective E. coli clones. Purified EpiD showed the typical absorption spectrum of an oxidized flavoprotein with maxima at 274, 382, and 453 nm. The coenzyme released from EpiD by heat treatment was identified as flavin mononucleotide. S. epidermidis Tü3298/EMS11, containing a mutation within epiD, was unable to synthesize active epidermin. This mutated gene, epiD*, was cloned in E. coli and expressed as an MBP-EpiD* fusion protein. DNA sequencing of epiD* identified a point mutation that led to replacement of Gly-93 with Asp. Unlike MBP-EpiD, the fusion protein MBP-EpiD* could not bind flavin mononucleotide. We propose that EpiD catalyzes the removal of two reducing equivalents from the cysteine residue of the C-terminal meso-lanthionine to form a --C==C-- double bond and is therefore involved in formation of the unusual S-[(Z)-2-aminovinyl[-D-cysteine structure in epidermin.