Purification of the polyenzymes responsible for tyrocidine synthesis and their dissociation into subunits

Lee, Sung G.; Roskoski, Robert; Bauer, Karl; Lipmann, Fritz

doi:10.1021/bi00727a006

Cited by 53 publications

(51 citation statements)

References 14 publications

(7 reference statements)

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“…For example, tyrocidine, a cyclic decapeptide antibiotic from Bacillus brevis, contains ornithine and three phenylalanines, two of which are in the D-configuration. The mechanism of nonribosomal peptide synthesis was most extensively investigated previously in the synthesis of tyrocidine (67)(68)(69)(70). Recently, the genes encoding the polyenzymes of tyrocidine synthesis have been cloned (71).…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Unique Glycosylated Anchor Endopeptidase That Cleaves the LPXTG Sequence Motif of Cell Surface Proteins of Gram-positive Bacteria

Lee

Pancholi

Fischetti

2002

Journal of Biological Chemistry

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The precursors of most surface proteins on Gram-positive bacteria have a C-terminal hydrophobic domain and charged tail, preceded by a conserved LPXTG motif that signals the anchoring process. This motif is the substrate for an enzyme, termed sortase, which has transpeptidation activity resulting in the cleavage of the LPXTG sequence and ultimate attachment of the protein to the peptidoglycan. While screening a group A streptococcal membrane extract for cleavage activity of the LPXTG motif, we identified an enzyme (which we term "LPXTGase") that differs significantly from sortase but also cleaves this motif. The enzyme is heavily glycosylated, which is required for its activity. Amino acid composition and sequence analysis revealed that LPXTGase differs from other enzymes, in that the molecule, which is about 14 kDa in size, has no aromatic amino acids, is rich in alanine, and is 30% composed of uncommon amino acids, suggesting a nonribosomal construction. A similar enzyme found in the membrane extract of Staphylococcus aureus, indicates that this unusual molecule may be common among Gram-positive bacteria. Whereas peptide antibiotics have been reported from bacillus species that also contain unusual amino acids and are synthesized non-ribosomally on amino acid-activating polyenzyme templates, this would be the first reported enzyme that may be similarly synthesized.A large group of cell surface proteins of Gram-positive bacteria are covalently anchored through their C termini to the cell wall peptidoglycan. Most of these proteins are essential for pathogenic bacteria to establish successful infection of host tissues, and hence they are considered virulence factors. Functionally, these surface proteins may be divided into three major groups: viz. 1) those with adhesin or invasion function (1-27); 2) those with antiphagocytic activities (28 -40); and 3) those that are enzymes that degrade surface components of host cells, thereby facilitating spread, and enzymes that hydrolyze large molecules in the surroundings into utilizable nutrients (29,(41)(42)(43)(44)(45)(46)(47)(48).A striking feature of all of these functionally and structurally diverse surface proteins is that they all possess a carboxylterminal LPXTG sequence (49), which is cleaved during surface translocation at the septum (50), resulting in a covalent linkage to cell wall peptidoglycan. In all cases, the genes for these proteins contain additional nucleotide sequences following that which encodes the LPXTG. These additional sequences encode a stretch of hydrophobic amino acids and positively charged C-terminal amino acids. Pancholi and Fischetti (51) observed that the hydrophobic and positively charged amino acid sequences are missing in the cell wall-linked M protein, indicating that the precursor of M protein was cleaved at a site within or immediately C-terminal to the LPXTG sequence. These findings strongly indicated that surface proteins become anchored to the cell wall by a common mechanism (49). Subsequently, it was shown that deletion of eith...

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Section: Discussionmentioning

confidence: 99%

Characterization of a Unique Glycosylated Anchor Endopeptidase That Cleaves the LPXTG Sequence Motif of Cell Surface Proteins of Gram-positive Bacteria

Lee

Pancholi

Fischetti

2002

Journal of Biological Chemistry

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“…shows strong similarity to a pantetheine-binding site (54), pantetheine was found only in TycB (29) and TycC (23) but not in TycA in a microbiological pantothenic acid assay (43). We used the in vivo incorporation of 3H-labeled 13-alanine to check the 4'-phosphopantetheine content of TycA.…”

Section: Incorporation Of P-[ H]alanine Although Core 6 Of Tycamentioning

confidence: 99%

“…Both compounds are produced nonribosomally by large multifunctional enzymes via the protein thiotemplate mechanism (32). The enzymes involved in the biosynthesis of linear gramicidin have not been completely characterized (2, 23), but the biosynthesis of tyrocidine has been studied in detail (26,27,29,30,49). The cyclic decapeptide tyrocidine (D-Phe-Pro-Phe-D-Phe-Asn-Glu-Tyr-ValOrn-Leu-)c is synthesized by the aid of three multifunctional enzymes; tyrocidine synthetases 1 (TycA), 2 (TycB), and 3 (TycC) activate and polymerize the 10 substrate amino acids.…”

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Analysis of core sequences in the D-Phe activating domain of the multifunctional peptide synthetase TycA by site-directed mutagenesis

1994

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The D-phenylalanine-activating enzyme tyrocidine synthetase I (TycA) from Bacilus brevis ATCC 8185 was overexpressed in Escherichia coli, purified to homogeneity, and assayed for ATP-PP. exchange and covalent binding of phenylalanine by the thiotemplate mechanism. Amino acid exchanges in four different cores of TycA created by site-directed mutagenesis revealed the amino acid residues involved in aminoacyladenylate formation and in covalent thioester formation. Mutations in the putative ATP-binding site SGTTGKPKG caused a decreased phenylalanine-dependent ATP-PP1 exchange activity to 10%v of the wild-type level for a Lys-186-to-Arg substitution and an almost complete loss of activity (<1%) for a Lys-186-to-Thr exchange. Under sporulation-inducing growth conditions, Bacillus brevis ATCC 8185 produces the peptide antibiotics tyrocidine and linear gramicidin (18). Both compounds are produced nonribosomally by large multifunctional enzymes via the protein thiotemplate mechanism (32). The enzymes involved in the biosynthesis of linear gramicidin have not been completely characterized (2, 23), but the biosynthesis of tyrocidine has been studied in detail (26,27,29,30,49). The cyclic decapeptide tyrocidine (D-Phe-Pro-Phe-D-Phe-Asn-Glu-Tyr-ValOrn-Leu-)c is synthesized by the aid of three multifunctional enzymes; tyrocidine synthetases 1 (TycA), 2 (TycB), and 3 (TycC) activate and polymerize the 10 substrate amino acids.TycA initiates peptide synthesis by activation of L-phenylalanine to the aminoacyladenylate by using ATP as the AMP donor. The activated phenylalanyladenylate is then covalently bound to TycA by a thioester linkage. After racemization, the D-isomer of phenylalanine is transferred from TycA to an activated proline residue on TycB. TycB activates proline and links it to two molecules of phenylalanine, while TycC, the third multienzyme, is responsible for the activation and condensation of the remaining six amino acids. Successive transpeptidation is achieved with the aid of the cofactor 4'-phosphopantetheine, which acts as an internal transport system for the growing peptide chain. To date, the presence of 4'-phosphopantetheine as a cofactor has been demonstrated for TycB and TycC (28, 30) but not for TycA. The cloning (34) and sequencing (64) of the tycA gene, however, revealed a putative 4'-phosphopantetheine-binding site similar to those found in grsB (58) and srfA (7,12 The tycA gene is located at the 5' end of the tyc operon (39), which is transcriptionally induced when cells enter the stationary phase of growth. Studies on the tyc promoter, which is located 330 bp upstream of the tycA initiation codon, revealed its dependence on the SpoOA-AbrB regulatory system (13,35,36,46). The nucleotide sequence of tycA codes for a protein of 1,087 residues with a calculated mass of 126 kDa.Sequence comparisons of TycA with other peptide synthetases and with a family of carboxy acid-activating enzymes revealed a high degree of similarity within a region of about 600 amino acids, which was designated the functional...

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“…Subsequently, activated amino acids are condensed stepwise in an aminoto carboxy-terminal direction. Previous investigations at the protein chemical level and, in particular, comparisons of several genes encoding peptide synthetases have revealed the modular architecture of this class of enzymes (4,23,28,42,44,53). One module is defined to harbor all catalytic activities to incorporate a single amino acid residue into the peptide product.…”

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The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains

1997

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The cyclic decapeptide antibiotic tyrocidine is produced by Bacillus brevis ATCC 8185 on an enzyme complex comprising three peptide synthetases, TycA, TycB, and TycC (tyrocidine synthetases 1, 2, and 3), via the nonribosomal pathway. However, previous molecular characterization of the tyrocidine synthetase-encoding operon was restricted to tycA, the gene that encodes the first one-module-bearing peptide synthetase. Here, we report the cloning and sequencing of the entire tyrocidine biosynthesis operon (39.5 kb) containing the tycA, tycB, and tycC genes. As deduced from the sequence data, TycB (404,562 Da) consists of three modules, including an epimerization domain, whereas TycC (723,577 Da) is composed of six modules and harbors a putative thioesterase domain at its C-terminal end. Each module incorporates one amino acid into the peptide product and can be further subdivided into domains responsible for substrate adenylation, thiolation, condensation, and epimerization (optional). We defined, cloned, and expressed in Escherichia coli five internal adenylation domains of TycB and TycC. Soluble His 6 -tagged proteins, ranging from 536 to 559 amino acids, were affinity purified and found to be active by amino acid-dependent ATP-PP i exchange assay. The detected amino acid specificities of the investigated domains manifested the colinear arrangement of the peptide product with the respective module in the corresponding peptide synthetases and explain the production of the four known naturally occurring tyrocidine variants. The K m values of the investigated adenylation domains for their amino acid substrates were found to be comparable to those published for undissected wild-type enzymes. These findings strongly support the functional integrities of single domains within multifunctional peptide synthetases. Directly downstream of the 3 end of the tycC gene, and probably transcribed in the tyrocidine operon, two tandem ABC transporters, which may be involved in conferring resistance against tyrocidine, and a putative thioesterase were found.The biosynthetic system required for the production of the cyclic decapeptide tyrocidine is one of the prototypes for a protein template-driven pathway to synthesize biologically active peptides by the nonribosomal mechanism (20,21,30,44,46). Large enzymes, called peptide synthetases, activate the amino acid constituents as aminoacyl adenylate at the expense of ATP and thioesterify them on the thiol moiety of an enzyme-attached cofactor, 4Ј-phosphopantetheine. Subsequently, activated amino acids are condensed stepwise in an aminoto carboxy-terminal direction. Previous investigations at the protein chemical level and, in particular, comparisons of several genes encoding peptide synthetases have revealed the modular architecture of this class of enzymes (4,23,28,42,44,53). One module is defined to harbor all catalytic activities to incorporate a single amino acid residue into the peptide product. The modules coincide in number with the number of incorporated amino acids and are arr...

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Purification of the polyenzymes responsible for tyrocidine synthesis and their dissociation into subunits

Cited by 53 publications

References 14 publications

Characterization of a Unique Glycosylated Anchor Endopeptidase That Cleaves the LPXTG Sequence Motif of Cell Surface Proteins of Gram-positive Bacteria

Characterization of a Unique Glycosylated Anchor Endopeptidase That Cleaves the LPXTG Sequence Motif of Cell Surface Proteins of Gram-positive Bacteria

Analysis of core sequences in the D-Phe activating domain of the multifunctional peptide synthetase TycA by site-directed mutagenesis

The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains

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