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.     

Analysis of genes involved in the biosynthesis of lantibiotic epidermin.

The structural gene of the lanthionine-containing peptide antibiotic epidermin is located on a 54-kb plasmid of Staphylococcus epidermidis [Schnell et al. (1988) Nature 333, 276-278]. A 13.5-kb DNA region neighbouring the epidermin structural gene (epiA) was subcloned and its sequencing revealed five additional open reading frames. Three of these reading frames, epiB, epiC and epiD shared no homology with previously described proteins stored in data bases. They were located 3' adjacent to epiA. Using epiB as a probe, a 5-kb mRNA was identified indicating that three or all four reading frames are transcribed as an operon. Additionally, a 0.3-kb mRNA specific for epiA was identified. Two open reading frames (epiP and epiQ) were located 3' to epiA, epiB, epiC and epiD, but in the reverse orientation. The epiQ gene product shows similarity to the positive regulatory factor PhoB. This might indicate a regulatory function of epiQ in epidermin biosynthesis. The epiP gene product shows striking similarity to several serine proteases which makes epiP a likely candidate for processing the epidermin prepeptide. Heterologous epidermin synthesis in the non-producing organism Staphylococcus carnosus finally proved that these reading frames are necessary for epidermin biosynthesis.     

Genetic analysis of epidermin biosynthetic genes and epidermin-negative mutants of Staphylococcus epidermidis.

Epidermin is produced by Staphylococcus epidermidis Tü3298 which harbors the 54-kb plasmid, pTü32. The plasmid contains not only the epidermin structural gene epiA, but also a flanking DNA region which is necessary for epidermin biosynthesis. The DNA sequence of this region revealed, in addition to epiA, five additional open reading frames, epiB, C, D, Q and P [Schnell, N., Engelke, G., Augustin J., Rosenstein, R., Ungermann, V., G?tz, F. & Entian, K.-D. (1992) Eur. J. Biochem. 204, 57-68]. We isolated a number of stable mutants from strain Tü3298 which are unable to produce biologically active epidermin. Complementation studies using the newly constructed staphylococcal plasmid vectors pT181mcs and pCU1 led to their classification as epiA, epiB, epiC or epiD mutants. Furthermore, evidence is presented that epiB lacks its own promoter and is co-transcribed from the epiA promoter. There is evidence that epiC and D possess their own promoters. Although epiQ and epiP mutants were not isolated, it could be shown by heterologous gene expression in S. carnosus and S. xylosus that the corresponding DNA region is involved in epidermin biosynthesis. We can not exclude the possibility that, in addition to the four open reading frames, epiA, B, C, D, and the DNA region comprising epiQ and P, host-encoded functions are necessary for epidermin production. Thus, the genetic information for epidermin biosynthesis in S. carnosus and S. xylosus is located on an 8-kb DNA fragment of pTü32. A further characterization of the two epiA mutants revealed that in both mutants, the preepidermin nucleotide sequence was changed. In one mutant, the mutation led to a substitution of Ser3 by Asn; in the other of Gly10 by Glu.     

Analysis of the Staphylococcus epidermidis genes epiF, -E, and -G involved in epidermin immunity.

The lantibiotic epidermin is produced by Staphylococcus epidermidis Tü3298. The known genes involved in epidermin biosynthesis and regulation are organized as operons (epiABCD and epiQP) that are encoded on the 54-kb plasmid pTü32. Here we describe the characterization of a DNA region that mediates immunity and increased epidermin production, located upstream of the structural gene epiA. The sequence of a 2.6-kb DNA fragment revealed three open reading frames, epiF, -E, and -G, which may form an operon. In the cloning host Staphylococcus carnosus, the three genes mediated an increased tolerance to epidermin, and the highest level of immunity (sevenfold) was achieved with S. carnosus carrying epiFEG and epiQ. The promoter of the first gene, epiF, responded to the activator protein EpiQ and contained a palindromic sequence similar to the EpiQ binding site of the epiA promoter, which is also activated by EpiQ. Inactivation of epiF, -E, or -G resulted in the complete loss of the immunity phenotype. An epidermin-sensitive S. epidermidis Tü3298 mutant was complemented by a DNA fragment containing all three genes. When the epiFEG genes were cloned together with plasmid pTepi14, containing the biosynthetic genes epiABCDQP, the level of epidermin production was approximately fivefold higher. The proteins EpiF, -E, and -G are similar in deduced sequence and proposed structure to the components of various ABC transporter systems. EpiF is a hydrophilic protein with conserved ATP-binding sites, while EpiE and -G have six alternating hydrophobic regions and very likely constitute the integral membrane domains. When EpiF was overproduced in S. carnosus, it was at least partially associated with the cytoplasmic membrane. A potential mechanism for how EpiFEG mediates immunity is discussed.     

Producer self-protection against the lantibiotic epidermin by the ABC transporter EpiFEG of Staphylococcus epidermidis Tü3298

Self-protection of the epidermin-producing strain Staphylococcus epidermidis Tü3298 against the pore-forming lantibiotic epidermin is mediated by an ABC transporter composed of the EpiF, EpiE, and EpiG proteins. We developed a sensitive assay based on HPLC analysis to investigate the capacity of the EpiFEG transporter to release epidermin and analogues from the cell surface to the external fluid. Our results indicate that the EpiFEG transporter works by expelling the lantibiotic from the cytoplasmic membrane into the surrounding medium. Analysis of transporter efficacy using nisin and gallidermin derivatives as substrates revealed a high substrate specificity. Furthermore, we showed that the activity of the gallidermin derivative L6G is enhanced by the presence of EpiE.     

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

Abstract 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.     

Overexpression, purification, and biochemical characterization of GumC, an enzyme involved in the biosynthesis of exopolysaccharide by Xylella fastidiosa

GumC is one of nine enzymes involved in the biosynthesis of fastidian gum, an exopolysaccharide produced by Xylella fastidiosa that may be linked directly to the pathogenicity of the microorganism. GumC may be responsible for gum polymerization or secretion through the membrane of X. fastidiosa. To perform structure and functions studies, we developed an expression system for the production of GumC as a fusion protein with maltose binding protein (MBP) using pMAL-c2x vector. The GumC-MBP fusion protein was expressed as a 94 kDa protein, which strongly reacts with anti-MBP antibodies. GumC-MBP was isolated by affinity chromatography through an amylose column and used to produce antibodies against the fusion protein. After the enzymatic cleavage of MBP, GumC was purified on a Q Sepharose Fast Flow column. GumC showed a molecular weight corresponding to the expected one (52 kDa) and its N-terminal sequence was identical to that deduced from the DNA. The shape of the circular dichroism spectrum was compatible with a folded protein that contains alpha-helical regions in its structure. Therefore, in this study we describe, for the first time, the production of GumC recombinant protein.     

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.     

Fermentation and isolation of epidermin,a lanthionine containing polypeptide antibiotic from Staphylococcus epidermidis

Summary Staphylococcus epidermidis TÜ 3298/DSM 3095 produces epidermin, a basic 21-residue peptide-amide antibiotic active against aerobic and anaerobic Gram-positive bacteria. Fermentations were performed by batch and feeding processes up to the 2001 scale. Highest yields were obtained when the first purification step was integrated into the fermentation process by on-line adsorption of the antibiotic. Isolation and purification by adsorption chromatography and ion exchange chromatography were performed batchwise.     

Overexpression,purification and characterization of SimL,an amide synthetase involved in simocyclinone biosynthesis

Simocyclinone D8 is a potent inhibitor of bacterial gyrase, produced by Streptomyces antibioticus Tü 6040. It contains an aminocoumarin moiety, similar to that of novobiocin, which is linked by an amide bond to a structurally complex acyl moiety, consisting of an aromatic angucycline polyketide nucleus, the deoxysugar olivose and a tetraene dicarboxylic acid. We have now investigated the enzyme SimL, responsible for the formation of the amide bond of simocyclinone. The gene was cloned, expressed in S. lividans T7, and the protein was purified to near homogeneity, and characterized. The 60 kDa protein catalyzed both the ATP-dependent activation of the acyl component as well as its transfer to the amino group of the aminocoumarin ring, with no requirement for a 4-phosphopantetheinyl cofactor. Besides its natural substrate, simocyclinone C4, SimL also accepted a range of cinnamic and benzoic acid derivatives and several other, structurally very diverse acids. These findings make SimL a possible tool for the creation of new aminocoumarin antibiotics.     

DnrD cyclase involved in the biosynthesis of doxorubicin: purification and characterization of the recombinant enzyme

Mutations in the Streptomyces peucetius dnrD gene block the ring cyclization leading from aklanonic acid methyl ester (AAME) to aklaviketone (AK), an intermediate in the biosynthetic pathway to daunorubicin (DNR) and doxorubicin. To investigate the role of DnrD in this transformation, its gene was overexpressed in Escherichia coli and the DnrD protein was purified to homogeneity and characterized. The enzyme was shown to catalyze the conversion of AAME to AK presumably via an intramolecular aldol condensation mechanism. In contrast to the analogous intramolecular aldol cyclization catalyzed by the TcmI protein from the tetracenomycin (TCM) C pathway in Streptomyces glaucescens, where a tricyclic anthraquinol carboxylic acid is converted to its fully aromatic tetracyclic form, the conversion catalyzed by DnrD occurs after anthraquinone formation and requires activation of a carboxylic acid group by esterification of aklanonic acid, the AAME precursor. Also, the cyclization is not coupled with a subsequent dehydration step that would result in an aromatic ring. As the substrates for the DnrD and TcmI enzymes are among the earliest isolable intermediates of aromatic polyketide biosynthesis, an understanding of the mechanism and active site topology of these proteins will allow one to determine the substrate and mechanistic parameters that are important for aromatic ring formation. In the future, these parameters may be able to be applied to some of the earlier polyketide cyclization processes that currently are difficult to study in vitro.     

Analysis of genes involved in biosynthesis of the lantibiotic subtilin.

Lantibiotics are peptide-derived antibiotics with high antimicrobial activity against pathogenic gram-positive bacteria. They are ribosomally synthesized and posttranslationally modified (N. Schnell, K.-D. Entian, U. Schneider, F. G?tz, H. Z?hner, R. Kellner, and G. Jung, Nature [London] 333:276-278, 1988). The most important lantibiotics are subtilin and the food preservative nisin, which both have a very similar structure. By using a hybridization probe specific for the structural gene of subtilin, spaS, the DNA region adjacent to spaS was isolated from Bacillus subtilis. Sequence analysis of a 4.9-kb fragment revealed several open reading frames with the same orientation as spaS. Downstream of spaS, no reading frames were present on the isolated XbaI fragment. Upstream of spaS, three reading frames, spaB, spaC, and spaT, were identified which showed strong homology to genes identified near the structural gene of the lantibiotic epidermin. The SpaT protein derived from the spaT sequence was homologous to hemolysin B of Escherichia coli, which indicated its possible function in subtilin transport. Gene deletions within spaB and spaC revealed subtilin-negative mutants, whereas spaT gene disruption mutants still produced subtilin. Remarkably, the spaT mutant colonies revealed a clumpy surface morphology on solid media. After growth on liquid media, spaT mutant cells agglutinated in the mid-logarithmic growth phase, forming longitudinal 3- to 10-fold-enlarged cells which aggregated. Aggregate formation preceded subtilin production and cells lost their viability, possibly as a result of intracellular subtilin accumulation. Our results clearly proved that reading frames spaB and spaC are essential for subtilin biosynthesis whereas spaT mutants are probably deficient in subtilin transport.     

Analysis of genes involved in biosynthesis of the lantibiotic subtilin.

Lantibiotics are peptide-derived antibiotics with high antimicrobial activity against pathogenic gram-positive bacteria. They are ribosomally synthesized and posttranslationally modified (N. Schnell, K.-D. Entian, U. Schneider, F. Götz, H. Zähner, R. Kellner, and G. Jung, Nature [London] 333:276-278, 1988). The most important lantibiotics are subtilin and the food preservative nisin, which both have a very similar structure. By using a hybridization probe specific for the structural gene of subtilin, spaS, the DNA region adjacent to spaS was isolated from Bacillus subtilis. Sequence analysis of a 4.9-kb fragment revealed several open reading frames with the same orientation as spaS. Downstream of spaS, no reading frames were present on the isolated XbaI fragment. Upstream of spaS, three reading frames, spaB, spaC, and spaT, were identified which showed strong homology to genes identified near the structural gene of the lantibiotic epidermin. The SpaT protein derived from the spaT sequence was homologous to hemolysin B of Escherichia coli, which indicated its possible function in subtilin transport. Gene deletions within spaB and spaC revealed subtilin-negative mutants, whereas spaT gene disruption mutants still produced subtilin. Remarkably, the spaT mutant colonies revealed a clumpy surface morphology on solid media. After growth on liquid media, spaT mutant cells agglutinated in the mid-logarithmic growth phase, forming longitudinal 3- to 10-fold-enlarged cells which aggregated. Aggregate formation preceded subtilin production and cells lost their viability, possibly as a result of intracellular subtilin accumulation. Our results clearly proved that reading frames spaB and spaC are essential for subtilin biosynthesis whereas spaT mutants are probably deficient in subtilin transport.     

Aspartate carbamoyltransferase from the thermoacidophilic archaeon Sulfolobus acidocaldarius. Cloning, sequence analysis, enzyme purification and characterization.

The genes coding for aspartate carbamoyltransferase (ATCase) in the extremely thermophilic archaeon Sulfolobus acidocaldarius have been cloned by complementation of a pyrBI deletion mutant of Escherichia coli. Sequencing revealed the existence of an enterobacterial-like pyrBI operon encoding a catalytic chain of 299 amino acids (34 kDa) and a regulatory chain of 170 amino acids (17.9 kDa). The deduced amino acid sequences of the pyrB and pyrI genes showed 27.6-50% identity with archaeal and enterobacterial ATCases. The recombinant S. acidocaldarius ATCase was purified to homogeneity, allowing the first detailed studies of an ATCase isolated from a thermophilic organism. The recombinant enzyme displayed the same properties as the ATCase synthesized in the native host. It is highly thermostable and exhibits Michaelian saturation kinetics for carbamoylphosphate (CP) and positive homotropic cooperative interactions for the binding of L-aspartate. Moreover, it is activated by nucleoside triphosphates whereas the catalytic subunits alone are inhibited. The holoenzyme purified from recombinant E. coli cells or present in crude extract of the native host have an Mr of 340 000 as estimated by gel filtration, suggesting that it has a quaternary structure similar to that of E. coli ATCase. Only monomers could be found in extracts of recombinant E. coli or Saccharomyces cerevisiae cells expressing the pyrB gene alone. In the presence of CP these monomers assembled into trimers. The stability of S. acidocaldarius ATCase and the allosteric properties of the enzyme are discussed in function of a modeling study.     

Expression, purification, and characterization of peptidyl-tRNA hydrolase from Staphylococcus aureus.

Bacterial peptidyl-tRNA hydrolase (Pth) activity ensures the rapid recycling of peptidyl-tRNAs that result from premature termination of translation. Pth has been shown to be essential for growth in Escherichia coli suggesting that its homologue in Staphylococcus aureus is a potential molecular therapeutic target for the development of antibacterial agents. In this report we describe the cloning of a DNA fragment (573 bp) containing the pth gene from a S. aureus (strain ISP3) genomic DNA library. Analysis of the predicted polypeptide sequence from the pth gene showed that the protein shared complete conservation of the three residues thought to be involved in the active site of E. coli Pth. The gene was cloned into a pQE-60 expression vector and expressed in E. coli, and the resulting His-tagged Pth protein was purified to greater than 95% purity from the soluble portion of the E. coli lysate in a single chromatographic step. His-tagged Pth was shown to be biologically active by its ability to hydrolyze diacetyl-[(3)H]Lys-tRNA(Lys) in a time- and concentration-dependent manner. Optimum hydrolyzing activity of Pth occurred at a pH value of 7.0 and a MgCl(2) concentration of 5 mM. The K(m) of the diacetyl-[(3)H]-Lys-tRNA(Lys) substrate for S. aureus Pth was determined to be 2.8 microM. A far UV circular dichroism spectrum revealed that His-tagged S. aureus Pth appears to have a structured core predominated by beta-sheet.     

Isolation and structural characterization of epilancin 15X, a novel lantibiotic from a clinical strain of Staphylococcus epidermidis

The potential application of lantibiotics as food-preserving agents and more recently as antibiotics has strongly increased the interest in these antibacterial peptides. Here, we report the elucidation of the primary and three-dimensional structures of the novel lantibiotic epilancin 15X from Staphylococcus epidermidis using high-resolution nuclear magnetic resonance spectroscopy and tandem mass spectrometry. The molecule contains ten post-translationally modified amino acids, three lanthionine ring structures and a hydroxy-propionyl N-terminal moiety. The primary and tertiary structure and the distribution of positive charges are closely similar to the previously identified lantibiotic epilancin K7, most likely indicative of a common mode of action.     

Structure prediction and functional analysis of KdsD, an enzyme involved in lipopolysaccharide biosynthesis

Lipopolysaccharide is an essential component of the outer membrane of Gram-negative bacteria and consists of three elements: lipid A, the core oligosaccharide and the O-antigen. The inner core region is highly conserved and contains at least one residue of 3-deoxy-d-manno-octulosonate (Kdo). The first committed step of Kdo biosynthesis is the aldol-keto isomerisation of d-ribulose 5-phosphate to d-arabinose 5-phosphate catalyzed by arabinose 5-phosphate isomerase encoded in Escherichia coli by the kdsD gene.KdsD contains an N-terminal sugar isomerase (SIS) domain commonly found in phosphosugar isomerases but its three-dimensional structure is unknown.The structure of the KdsD SIS domain has been predicted by homology modeling using the hypothetical 3etn protein as a template. Moreover by sequence alignments, comparison with other sugar isomerases structurally related to KdsD, and site-directed mutagenesis we implicated four residues in KdsD activity or substrate recognition. A possible role of these residues in the catalysis is discussed.     

Expression,purification, and characterization of AknX anthrone oxygenase,which is involved in aklavinone biosynthesis in Streptomyces galilaeus

In streptomycete anthracycline biosynthetic gene clusters, small open reading frames are located just upstream of minimal polyketide synthase genes. aknX is such a gene found in the aklavinone-aclacinomycin biosynthetic gene cluster of Streptomyces galilaeus. In order to identify its function, the aknX gene was expressed in Escherichia coli. The cell extract prepared from E. coli cells overexpressing AknX protein exhibited anthrone oxygenase activity, which converted emodinanthrone to anthraquinone emodin. This indicates that AknX and related gene products such as DnrG and SnoaB are involved in the formation of aklanonic acid from its anthrone precursor, as suggested by their homology with TcmH and ActVA6. The AknX protein fused with a His(6) tag was efficiently purified to homogeneity by Ni(2+) affinity and anion-exchange column chromatography. The native molecular mass of AknX was estimated to be 42 kDa by gel filtration. Thus, native AknX is considered to have a homotrimeric subunit structure. AknX, like TcmH and ActVA6, possesses no apparent prosthetic group for oxygen activation. Site-directed mutagenesis was carried out to identify the key amino acid residue(s) involved in the oxygenation reaction. Of seven AknX mutants expressed, the W67F mutant showed significantly reduced oxygenase activity, suggesting the important role of the W67 residue in the AknX reaction. A possible mechanism for the reaction via peroxy anion intermediate is proposed.     

Partial purification and characterization of an enzyme involved in the formation of beta-aspartyl dipeptides in rat kidney.

The formation of beta-aspartyl-glycine from asparagine and glycine was demonstrated in the supernatant of rat kidney. The enzyme involved in this process was partially purified. Based on the properties of the enzyme reaction and the coincidence of purification rates of this activity and asparaginase, it can be speculated that the enzyme is a kind of asparaginase. Examination of the preference for beta-aspartyl donors and acceptors showed that asparagine and glycine were the preferred donor and acceptor, respectively. beta-Aspartyl dipeptides also transferred their aspartyl residues to amino acids. Amino acids other than glycine also accepted the aspartyl moiety from the donors.     

Overexpression, purification, and characterization of isochorismate synthase (EntC), the first enzyme involved in the biosynthesis of enterobactin from chorismate

Isochorismate synthase (EC 5.4.99.6), the entC gene product of Escherichia coli, catalyzes the conversion of chorismate to isochorismate, the first step in the biosynthesis of the powerful iron-chelating agent enterobactin. A sequence-specific deletion method has been used to construct an EntC overproducer, which allows for the purification and characterization of the E. coli isochorismate synthase for the first time. The N-terminal sequence and the subunit molecular weight (43,000) of the polypeptide derived from SDS-polyacrylamide gel electrophoresis agree with those deduced from DNA sequence data. The enzyme is an active monomer with a native molecular weight of 42,000. It was shown that EntC alone is fully capable of catalyzing the interconversion of chorismate and isochorismate in both directions and the associated activity is not affected by EntA of the same biosynthetic pathway as has recently been speculated [Elkins, M. F., & Earhart, C. F. (1988) FEMS Microbiol. Lett. 56, 35; Liu, J., Duncan, K., & Walsh, C.T. (1989) J. Bacteriol. 171, 791; Ozenberger, B. A., Brickman, T.J., & McIntosh, M. A. (1989) J. Bacteriol. 171, 775]. The kinetic constants were determined with Km = 14 microM and kcat = 173 min-1 for chorismate in the forward direction and Km = 5 microM and kcat = 108 min-1 for isochorismate in the backward direction. The equilibrium constant for the reaction derived from the kinetic data is 0.56 with the equilibrium lying toward the side of chorismate, corresponding to a free energy difference of 0.36 kcal/mol between chorismate and isochorismate.(ABSTRACT TRUNCATED AT 250 WORDS)