Activation of the Pacidamycin PacL Adenylation Domain by MbtH-like Proteins

Zhang, Wenjun; Heemstra, John R.; Walsh, Christopher T.; Imker, H.J.

doi:10.1021/bi101539b

Cited by 86 publications

(113 citation statements)

References 11 publications

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“…As shown in a phylogenetic analysis of MbtH-like proteins by Zhang et al (7), CdaX and CloY are situated in close proximity in one branch of the phylogenetic tree, and Orf1van and SimY in another branch. The sequence identity among these proteins is ϳ60%.…”

Section: Expression and Purification Of Tyrosine-adenylating Enzymes mentioning

confidence: 87%

“…The three-dimensional structures of two MbtH-like proteins have been experimentally determined (4,5), but again this did not allow their function to be determined. The first biochemical evidence for the function of MbtH-like proteins in non-ribosomal peptide biosynthesis has been recently provided in two rapid reports by Felnagle et al (6) and Zhang et al (7). Additional data were presented as part of a study on glidobactin biosynthesis (8).…”

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confidence: 99%

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Role of MbtH-like Proteins in the Adenylation of Tyrosine during Aminocoumarin and Vancomycin Biosynthesis

Boll

Taubitz

Heide

2011

Journal of Biological Chemistry

109

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Background: L-Tyrosine adenylation is a key step in aminocoumarin antibiotic and vancomycin biosynthesis. Results: Several but not all tyrosine-adenylating enzymes require MbtH-like proteins for activity, forming heterotetrameric complexes. A single Leu-to-Met mutation creates an MbtH-independent enzyme. Conclusion and Significance: MbtH-like proteins are essential tools in the combinatorial biosynthesis of antibiotics.

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Section: Expression and Purification Of Tyrosine-adenylating Enzymes mentioning

confidence: 87%

mentioning

confidence: 99%

Role of MbtH-like Proteins in the Adenylation of Tyrosine during Aminocoumarin and Vancomycin Biosynthesis

Boll

Taubitz

Heide

2011

Journal of Biological Chemistry

109

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“…2A and Table 1). PyrH (71 aa) is 74% identical to the MbtH-like protein from Streptomyces fungicidicus, and it contains the three conserved Trp residues that may be important for moderating protein-protein interactions (42,43). Similar proteins are integral parts of several NRPSs that stimulate specific A domains (44).…”

Section: Unknown or Tentative Role In Pyridomycin Biosynthesis-mentioning

confidence: 99%

Identification and Characterization of the Pyridomycin Biosynthetic Gene Cluster of Streptomyces pyridomyceticus NRRL B-2517

Huang

Wang

Yin

et al. 2011

Journal of Biological Chemistry

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Pyridomycin is a structurally unique antimycobacterial cyclodepsipeptide containing rare 3-(3-pyridyl)-L-alanine and 2-hydroxy-3-methylpent-2-enoic acid moieties. The biosynthetic gene cluster for pyridomycin has been cloned and identified from Streptomyces pyridomyceticus NRRL B-2517. Sequence analysis of a 42.5-kb DNA region revealed 26 putative open reading frames, including two nonribosomal peptide synthetase (NRPS) genes and a polyketide synthase gene. A special feature is the presence of a polyketide synthase-type ketoreductase domain embedded in an NRPS. Furthermore, we showed that PyrA functioned as an NRPS adenylation domain that activates 3-hydroxypicolinic acid and transfers it to a discrete peptidyl carrier protein, PyrU, which functions as a loading module that initiates pyridomycin biosynthesis in vivo and in vitro. PyrA could also activate other aromatic acids, generating three pyridomycin analogues in vivo.

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“…Recently we (7) and others (17,49) determined that members of the MbtH-like protein superfamily are small proteins that influence the solubility and function of associated NRPS compo- nents. One of the most important proposals from these studies is that NRPSs that were previously recalcitrant to in vitro analysis due to insolubility or inactivity issues may become soluble and active when coproduced with their cognate MbtH-like protein.…”

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confidence: 99%

Analyses of MbtB, MbtE, and MbtF Suggest Revisions to the Mycobactin Biosynthesis Pathway in Mycobacterium tuberculosis

2012

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The production of mycobactin (MBT) by Mycobacterium tuberculosis is essential for this bacterium to access iron when it is in an infected host. Due to this essential function, there is considerable interest in deciphering the mechanism of MBT assembly, with the goal of targeting select biosynthetic steps for antituberculosis drug development. The proposed scheme for MBT biosynthesis involves assembly of the MBT backbone by a hybrid nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) megasynthase followed by the tailoring of this backbone by N 6 acylation of the central l -Lys residue and subsequent N 6 -hydroxylation of the central N 6 -acyl- l -Lys and the terminal caprolactam. A complete testing of this hypothesis has been hindered by the inability to heterologously produce soluble megasynthase components. Here we show that soluble forms of the NRPS components MbtB, MbtE, and MbtF are obtained when these enzymes are coproduced with MbtH. Using these soluble enzymes we determined the amino acid specificity of each adenylation (A) domain. These results suggest that the proposed tailoring enzymes are actually involved in precursor biosynthesis since the A domains of MbtE and MbtF are specific for N 6 -acyl- N 6 -hydroxy- l -Lys and N 6 -hydroxy- l -Lys, respectively. Furthermore, the preference of the A domain of MbtB for l -Thr over l -Ser suggests that the megasynthase produces MBT derivatives with β-methyl oxazoline rings. Since the most prominent form of MBT produced by M. tuberculosis lacks this β-methyl group, a mechanism for demethylation remains to be discovered. These results suggest revisions to the MBT biosynthesis pathway while also identifying new targets for antituberculosis drug development.

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Activation of the Pacidamycin PacL Adenylation Domain by MbtH-like Proteins

Cited by 86 publications

References 11 publications

Role of MbtH-like Proteins in the Adenylation of Tyrosine during Aminocoumarin and Vancomycin Biosynthesis

Role of MbtH-like Proteins in the Adenylation of Tyrosine during Aminocoumarin and Vancomycin Biosynthesis

Identification and Characterization of the Pyridomycin Biosynthetic Gene Cluster of Streptomyces pyridomyceticus NRRL B-2517

Analyses of MbtB, MbtE, and MbtF Suggest Revisions to the Mycobactin Biosynthesis Pathway in Mycobacterium tuberculosis

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