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

Boll, Björn; Taubitz, Tatjana; Heide, Lutz

doi:10.1074/jbc.m111.288092

Cited by 62 publications

(115 citation statements)

References 31 publications

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“…This amino acid composition was confirmed by all three amino acid prediction tools used by antiSMASH 2.0 (7,36). Hence, the assembled heptapeptide has the predicted amino acid sequence tions (37,38,39,40). The predicted incorporation of aromatic amino acids into the heptapeptide revealed that the A. japonicum glycopeptide belongs to either the type II, type III, or type IV glycopeptides but not to the type I (vancomycin type) glycopeptides characterized by aliphatic amino acids at positions 1 and 3 (Fig.…”

Section: Resultsmentioning

confidence: 73%

Overproduction of Ristomycin A by Activation of a Silent Gene Cluster in Amycolatopsis japonicum MG417-CF17

Spohn

Kirchner

Kulik

et al. 2014

Antimicrob Agents Chemother

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eThe emergence of antibiotic-resistant pathogenic bacteria within the last decades is one reason for the urgent need for new antibacterial agents. A strategy to discover new anti-infective compounds is the evaluation of the genetic capacity of secondary metabolite producers and the activation of cryptic gene clusters (genome mining). One genus known for its potential to synthesize medically important products is Amycolatopsis. However, Amycolatopsis japonicum does not produce an antibiotic under standard laboratory conditions. In contrast to most Amycolatopsis strains, A. japonicum is genetically tractable with different methods. In order to activate a possible silent glycopeptide cluster, we introduced a gene encoding the transcriptional activator of balhimycin biosynthesis, the bbr gene from Amycolatopsis balhimycina (bbr Aba ), into A. japonicum. This resulted in the production of an antibiotically active compound. Following whole-genome sequencing of A. japonicum, 29 cryptic gene clusters were identified by genome mining. One of these gene clusters is a putative glycopeptide biosynthesis gene cluster. Using bioinformatic tools, ristomycin (syn. ristocetin), a type III glycopeptide, which has antibacterial activity and which is used for the diagnosis of von Willebrand disease and Bernard-Soulier syndrome, was deduced as a possible product of the gene cluster. Chemical analyses by high-performance liquid chromatography and mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) spectroscopy confirmed the in silico prediction that the recombinant A. japonicum/pRM4-bbr Aba synthesizes ristomycin A.

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Section: Resultsmentioning

confidence: 73%

Overproduction of Ristomycin A by Activation of a Silent Gene Cluster in Amycolatopsis japonicum MG417-CF17

Spohn

Kirchner

Kulik

et al. 2014

Antimicrob Agents Chemother

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“…Expression, Purification, and Biochemical Investigation of SlgN1-We have recently shown that YbdZ, an MbtH-like protein encoded in the genome of E. coli, forms complexes with adenylating enzymes when these are expressed in E. coli, obscuring their true biochemical properties (11). Therefore, we expressed all SlgN1 proteins in this study in a previously developed E. coli strain that lacks the ybdZ gene (11).…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, we expressed all SlgN1 proteins in this study in a previously developed E. coli strain that lacks the ybdZ gene (11). SlgN1 was initially expressed as an N-terminally His-tagged protein and investigated for its adenylating activity using a pyrophosphate exchange assay following the procedure described in a previous study (11).…”

Section: Resultsmentioning

confidence: 99%

Structural Basis of the Interaction of MbtH-like Proteins, Putative Regulators of Nonribosomal Peptide Biosynthesis, with Adenylating Enzymes

Herbst

Boll

Zocher

et al. 2013

Journal of Biological Chemistry

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Background: MbtH-like proteins are required for many adenylation reactions in nonribosomal peptide biosynthesis. Results: We present the crystal structure of the adenylating enzyme SlgN1, involved in the biosynthesis of the antibiotic streptolydigin, and analyze its interface with an MbtH-like domain. Conclusion: and Ala-433 (adenylation domain) are required for domain interaction. Significance: MbtH-like proteins activate adenylating enzymes but make no direct contact with their substrates.

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“…Adenylation domains can require MLPs for either solubility or activity, and some, such as EntF and NovH (11), are unusual as they function without MLPs but show increased activity upon MLP binding. We asked whether these structural and biochemical data could allow one to determine from an amino acid sequence if an adenylation domain will accommodate an MLP.…”

Section: Discussionmentioning

confidence: 99%

“…Some adenylation domains also require MLPs as chaperones and cannot be expressed without their MLP partner (10). Furthermore, genetic and biochemical studies have shown that MLPs can activate NRPS proteins in different biosynthetic clusters within a species and can be substituted heterologously in different species (11).…”

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confidence: 99%

Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture

Miller

Drake

Shi

et al. 2016

Journal of Biological Chemistry

100

122

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Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein, an amino acidloading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might be derived from understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full-length NRPS EntF bound to the MLPs from Escherichia coli and Pseudomonas aeruginosa. These new structures, along with biochemical and bioinformatics support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally, the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain. Nonribosomal peptide synthetases (NRPSs)2 are fascinating modular enzymes that use an assembly line architecture to produce important peptide natural products (1-3). During synthesis, the amino acid building blocks are bound to peptidyl carrier protein (PCP) domains that migrate between catalytic active sites for the requisite steps in the biosynthetic pathway. Most NRPS modules contain an adenylation domain that activates the correct amino acid and loads the PCP domain. Internal modules contain a condensation domain that transfers the upstream amino acid or peptide to the newly loaded amino acid, extending the peptide length by one residue. Freed from the constraints of standard ribosomal synthesis, NRPS products display a wide range of chemical structures.The chemical diversity of NRPS products is further enhanced by the presence of additional internal domains or external proteins that modify the nascent peptide (4 -6). In addition to these tailoring enzymes, some NRPS biosynthetic clusters contain genes encoding small (ϳ70-residue) proteins. Named after the MbtH protein from the Mycobacterium tuberculosis mycobactin operon (7), these MbtH-like proteins (MLPs) were shown simultaneously by Thomas and co-workers (8) and Walsh and co-workers (9) to function as activators of acyladenylate formation. The MLP-NRPS interactions exhibit several interesting features. Some adenylation domains also require MLPs as chaperones and cannot be expressed without their MLP partner (10). Furthermore, genetic and biochemical studies have shown that MLPs can activate NRPS proteins in different biosynthetic clusters within a species and can be substituted heterologously in different species (11).Structural studies of MLPs and MLP-adenylation d...

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

Cited by 62 publications

References 31 publications

Overproduction of Ristomycin A by Activation of a Silent Gene Cluster in Amycolatopsis japonicum MG417-CF17

Overproduction of Ristomycin A by Activation of a Silent Gene Cluster in Amycolatopsis japonicum MG417-CF17

Structural Basis of the Interaction of MbtH-like Proteins, Putative Regulators of Nonribosomal Peptide Biosynthesis, with Adenylating Enzymes

Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture

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