A Domain of RubC1 of Rubradirin Biosynthesis Can Functionally Replace MbtH‐Like Proteins in Tyrosine Adenylation

Boll, Björn; Heide, Lutz

doi:10.1002/cbic.201200633

Cited by 5 publications

(3 citation statements)

References 8 publications

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“…32 They seem to have high specicity for their particular A domain partners, but some examples of weak complementarity are reported. 33 The roles of MbtH-like proteins have been explored in a variety of biochemical and structural studies, 17,[33][34][35][36][37][38][39][40][41][42][43][44][45][46] but their exact functions and mechanisms of action still remain elusive.…”

Section: Adenylation Domain Core Signature Sequences and Structuresmentioning

confidence: 99%

Interrupted adenylation domains: unique bifunctional enzymes involved in nonribosomal peptide biosynthesis

2015

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Nonribosomal peptides (NRPs) account for a large portion of drugs and drug leads currently available in the pharmaceutical industry. They are one of two main families of natural products biosynthesized on megaenzyme assembly-lines composed of multiple modules that are, in general, each comprised of three core domains and on occasion of accompanying auxiliary domains. The core adenylation (A) domains are known to delineate the identity of the specific chemical components to be incorporated into the growing NRPs. Previously believed to be inactive, A domains interrupted by auxiliary enzymes have recently been proven to be active and capable of performing two distinct chemical reactions. This highlight summarizes current knowledge on A domains and presents the various interrupted A domains found in a number of nonribosomal peptide synthetase (NRPS) assembly-lines, their predicted or proven dual functions, and their potential for manipulation and engineering for chemoenzymatic synthesis of new pharmaceutical agents with increased potency.

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Section: Adenylation Domain Core Signature Sequences and Structuresmentioning

confidence: 99%

Interrupted adenylation domains: unique bifunctional enzymes involved in nonribosomal peptide biosynthesis

2015

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“…The full genome of S. sibiricum is not yet available, and hence whether there is an MLP‐encoding gene elsewhere in the genome is not known. In some examples, MLPs from certain hosts can replace the function of MLPs from native sources . For instance, the MLPs CloY and SimY coded by gene clusters from different Streptomyces species responsible for the biosynthesis of chlorobiocin and simocyclinone D8, respectively, can activate both of the tyrosine adenylases that initiate the assembly of a common aminocoumarin moiety .…”

Section: Methodsmentioning

confidence: 99%

An Activator of an Adenylation Domain Revealed by Activity but Not Sequence Homology

Saha

Rokita

2016

ChemBioChem

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Nonribosomal peptide synthetases (NRPSs), responsible for synthesizing many medicinally important natural products, frequently use adenylation domain activators (ADAs) to promote substrate loading. Although ADAs are usually MbtH-like proteins (MLPs), a new type of ADA appears to promote an NRPS-dependent incorporation of a dihydropyrrole unit into sibiromycin. The adenylation and thiolation didomain of the NRPS SibD catalyzes the adenylation of a limited number of amino acids including L-tyr, the precursor in dihydropyrrole biosynthesis, using a standard radioactivity exchange assay. LC-MS/MS analysis confirmed loading of L-tyr onto the thiolation domain. SibB, a small protein with no prior functional assignment nor sequence homology to MLPs, was found to promote the exchange activity. MLPs from bacteria expressing homologous biosynthetic pathways were unable to replace this function of SibB. Discovery of this new type of ADA demonstrates the importance of searching beyond the conventional MLP standard for proteins affecting NRPS activity. Graphical AbstractNonribosomal peptide synthetases (NRPSs) frequently use MbtH-like proteins to promote activity of their adenylation domains. Sibiromycin biosynthesis provides an example of a pathway that lacks such a protein and instead uses an atypical adenylation domain activator, SibB, to promote the activity of the NRPS SibD.Correspondence to: Shalini Saha; Steven E. Rokita. Experimental SectionAll experimental details including the construction of expression vectors, production and purification of proteins, synthesis and characterization of all compounds, and assays for radioisotope exchange and NRPS acylation are described in supporting information. Elongation modules include an additional condensation (C) domain for coupling two NRPSconjugates through a peptide bond. Termination modules contain yet another domain most typically a thioesterase (TE) domain or infrequently a reductase (R) domain that promotes hydrolysis of the nascent peptide from the NRPS. These modules often are complemented by a protein known as an A domain activator (ADA). [2] ADAs are small proteins that stimulate A domain activity and were first found necessary for successful biosynthesis of capreomycin and viomycin. [3] In a few cases, NRPSs were not even observed or isolated in a soluble form after heterologous expression without coexpression of an appropriate ADA. [4] The first ADAs reported were all MbtH-like proteins (MLPs) named after a homolog (MbtH) in Mycobacterium tuberculosis and identifiable by a Pfam domain (PF03621). [5] More recently, two new ADAs have been discovered that are devoid of the MLP signature sequences. One of these resembles an incomplete C domain and the other shares very low sequence similarity to known NRPSs (E > 0.002). [6] Both of these ADAs as well as one example of an MLP are covalently fused to their A domains. [6][7] In contrast, the vast majority of ADAs exist as independent proteins encoded by genes within their associated biosynthetic gene cluster...

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“…While most MLPs are independent proteins, some are covalently joined to adenylation domains in multidomain proteins. Additional adenylation domain activators have also been observed in other multidomain NRPSs [40,41]. Finally, other domains have been identified that serve as scaffolds for the interaction with additional catalytic partners.…”

Section: Combining Nrps Domains To Build a Modulementioning

confidence: 99%

Structural insight into the necessary conformational changes of modular nonribosomal peptide synthetases

Gulick

2016

Current Opinion in Chemical Biology

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Nonribosomal peptide synthetases (NRPSs) catalyze the assembly line biosynthesis of peptide natural products that play important roles in microbial signaling and communication. These multidomain enzymes use an integrated carrier protein that delivers the growing peptide to the catalytic domains, requiring coordinated conformational changes that allow the proper sequence of synthetic steps. Recent structural studies of NRPSs have described important conformational states and illustrate the critical role of a small subdomain within the adenylation domains. This subdomain alternates between catalytic conformations and also serves as a linker domain, providing further conformational flexibility to enable the carrier to project from the core of NRPS. These studies are described along with remaining questions in the study of the structural dynamics of NRPSs.

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A Domain of RubC1 of Rubradirin Biosynthesis Can Functionally Replace MbtH‐Like Proteins in Tyrosine Adenylation

Abstract: Master of your domain: A domain of the bifunctional enzyme RubC1 catalyzes the adenylation of tyrosine. This domain can be activated not only by MbtH-like proteins but also by another domain of RubC1 that has no similarity to MbtH-like proteins.

Cited by 5 publications

References 8 publications

Interrupted adenylation domains: unique bifunctional enzymes involved in nonribosomal peptide biosynthesis

Interrupted adenylation domains: unique bifunctional enzymes involved in nonribosomal peptide biosynthesis

An Activator of an Adenylation Domain Revealed by Activity but Not Sequence Homology

Structural insight into the necessary conformational changes of modular nonribosomal peptide synthetases

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