Identification of a Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin

Mycobacterium tuberculosis (Mt) produces complex virulence-enhancing lipids with scaffolds consisting of phthiocerol and phthiodiolone dimycocerosate esters (PDIMs). Sequence analysis suggested that PapA5, a so-called polyketide-associated protein (Pap) encoded in the PDIM synthesis gene cluster, as well as PapA5 homologs found in Mt and other species, are a subfamily of acyltransferases. Studies with recombinant protein confirmed that PapA5 is an acetyltransferase. Deletion analysis in Mt demonstrated that papA5 is required for PDIM synthesis. We propose that PapA5 catalyzes diesterification of phthiocerol and phthiodiolone with mycocerosate. These studies present the functional characterization of a Pap and permit inferences regarding roles of other Paps in the synthesis of complex lipids, including the antibiotic rifamycin

show abstract

“…His-6-tagged Pseudomonas aeruginosa arylation enzyme PchD and His-6-tagged Mt synthetase MbtB ArCP domain were purified as reported (26,27).…”

Section: Methodsmentioning

confidence: 99%

Mycobacterial polyketide-associated proteins are acyltransferases: Proof of principle with Mycobacterium tuberculosis PapA5

Onwueme

Ferreras

Buglino

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…First attempts to excise and express the EntF A domain as a typical 50-to 60-kDa A domain fragment failed to give soluble folded protein with any detectable activity (23), highlighting the ongoing unpredictability of stable folding of internal NRPS domains (26,(32)(33)(34). Therefore, we retained the native N terminus of EntF and expressed the two domain C-A fragment of EntF as a 108-kDa protein (residues 1-974), which proved to be soluble and contain an active A domain when purified as a C-terminal His 6 -tagged fragment.…”

Section: Discussionmentioning

confidence: 99%

The EntF and EntE adenylation domains of Escherichia coli enterobactin synthetase: Sequestration and selectivity in acyl-AMP transfers to thiolation domain cosubstrates

Ehmann

Shaw-Reid

Losey

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

116

101

View full text Add to dashboard Cite

Enterobactin, the tris-(N-(2,3-dihydroxybenzoyl)serine) trilactone siderophore of Escherichia coli, is synthesized by a three-protein (EntE, B, F) six-module nonribosomal peptide synthetase (NRPS). In this work, the 142-kDa four-domain protein EntF was bisected into two double-domain fragments: a 108-kDa condensation and adenylation construct, EntF C-A, and a 37-kDa peptidyl carrier protein (PCP) and thioesterase protein, EntF PCP-TE. The adenylation domain activity of EntF C-A formed seryl-AMP but lost the ability to transfer the seryl moiety to the cognate EntF PCP-TE in trans. Seryl transfer to heterologous PCP protein fragments, the SrfB1 PCP from surfactin synthetase and Ybt PCP1 from yersiniabactin synthetase, was observed at rates of 0.5 min ؊1 and 0.01 min ؊1 , respectively. The possibility that these slow acylation rates reflected dissociation of acyl͞aminoacyl-AMP followed by adventitious thiolation by the heterologous PCPs in solution was addressed by measuring catalytic turnover of pyrophosphate (PPi) released from the adenylation domain. The holo SrfB1 PCP protein as well as Ybt PCP1 did not stimulate an increase in PPi release from EntF C-A or EntE. In this light, aminoacylations in trans between A and PCP domain fragments of NRPS assembly lines must be subjected to kinetic scrutiny to determine whether transfer is truly between protein domains or results from slow aminoacyl-AMP release and subsequent nonenzymatic thiol capture. N onribosomal peptide synthetases (NRPS) activate a broad range of amino acids, including numerous nonproteinogenic amino acids (1, 2) and incorporate them into growing peptidyl chains as an elongating series of peptidyl-S-enzyme intermediates. This is an RNA-independent templated chaingrowth process with an assembly line organization of different catalytic and carrier protein domains whose placement and function determine the length and structure of the released peptide natural product. Both the amino acid monomers and the elongating peptide chains are tethered as thioester intermediates (acyl-S-enzymes) to the terminal thiol of phosphopantetheinyl (Ppant) moieties of peptidyl carrier protein (PCP) domains, also known as thiolation (T) domains for their functions as thioltethering way stations. The Ppant thiol in the PCP domains is added posttranslationally by priming enzymes (3, 4) to a conserved serine side chain in each PCP domain, converting it from inactive apo form to holo form capable of aminoacylation.The 8-to 10-kDa peptidyl chain-carrying PCP domains are interspersed among and paired with two catalytic domains that make up the core of each NRPS elongation module: 50-to 60-kDa adenylation (A) domains for each amino acid to be activated and 50-to 60-kDa condensation (C) domains for each peptide bond-forming chain-translocating step. Thus a typical NRPS module consists of the core triad of (C-A-PCP) n domains as the functional unit competent for: selection and activation of a given amino acid as the aminoacyl-AMP (Fig. 1, Step 1), covalent loading of the aminoacyl m...

show abstract

“…silvaticum, as well as from M. intracellulare, M. malmoense, M. tuberculosis H37Rv, M. phlei, M. fortuitum and M. smegmatis. The deleted region included the locus 6 flanking genes consisting of a homologue (38 % identity) to the polyketide synthase pks17 : Rv1663 of M. tuberculosis (Quadri et al, 1998), and a homologue (38 % identity) of a transcriptional regulator, tetR, from S. coelicolor (Marvaud et al, 1998). The loss of this locus, however, is inconsistent with both MPIL M1 and M4 profiles exhibiting similar BstEII RFLP C1 profiles (Table 3).…”

Section: Discussionmentioning

confidence: 99%

Characterization of IS900 loci in Mycobacterium avium subsp. paratuberculosis and development of multiplex PCR typing The GenBank accession numbers for the sequences reported in this paper are AJ011838, AJ250015–AJ250023 and AJ251434–AJ251437.

et al. 2000

View full text Add to dashboard Cite

Mycobacterium avium subsp. paratuberculosis is a pathogen that causes chronic inflammation of the intestine in many animals, including primates, and is implicated in Crohn's disease in humans. It differs from other members of the M. avium complex in having 14-18 copies of IS900 inserted into conserved loci in its genome. In the present study, genomic DNA flanking 14 of these insertions was characterized and homologues in the Mycobacterium tuberculosis and M. avium subsp. avium genomes were identified. These included regions encoding a sigma factor (sigJ) at locus 3, a nitrate reductase (nirA) at locus 4, a transcription regulator (tetR) and polyketide synthase at locus 6, and a 6-O-methylguanine methyltransferase at locus 9. In addition, locus numbers were assigned to 9 of 15 RFLP bands previously described. IS900 insertion at 7 of the 14 characterized loci was into the RBS of a gene substituting an RBS encoded by IS900 sited two bases closer to the initiation codon. IS900 insertion at five loci interrupted an ORF at the target site, one of which encoded a homologue of the immunodominant mycobacterial DesA1 protein. Eleven of eighty-one M. avium subsp. paratuberculosis isolates lacked the insertion site at locus 6 together with flanking genomic DNA. This region was also absent from seven reference strains of M. avium subsp. avium, from one M. avium subsp. silvaticum and from six other mycobacterial species. A multiplex PCR of IS900 loci (MPIL) typing method was developed which was able to discriminate 10 different types of M. avium subsp. paratuberculosis from the panel of 81 isolates with consistent differences between those of bovine and ovine origin. Nine MPIL types corresponded with a single PstI/BstEII RFLP type, suggesting that this method may be applicable to typing of M. avium subsp. paratuberculosis directly from a sample without the need for culture. The remaining MPIL type corresponded with seven PstI/BstEII RFLP types. Further resolution of these may come from sequencing the remaining four uncharacterized IS900 loci.

show abstract

Identification of a Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin

Cited by 412 publications

References 34 publications

Mycobacterial polyketide-associated proteins are acyltransferases: Proof of principle with Mycobacterium tuberculosis PapA5

Mycobacterial polyketide-associated proteins are acyltransferases: Proof of principle with Mycobacterium tuberculosis PapA5

The EntF and EntE adenylation domains of Escherichia coli enterobactin synthetase: Sequestration and selectivity in acyl-AMP transfers to thiolation domain cosubstrates

Characterization of IS900 loci in Mycobacterium avium subsp. paratuberculosis and development of multiplex PCR typing The GenBank accession numbers for the sequences reported in this paper are AJ011838, AJ250015–AJ250023 and AJ251434–AJ251437.

Contact Info

Product

Resources

About