An Engineered Lantibiotic Synthetase That Does Not Require a Leader Peptide on Its Substrate

Oman, Trent J.; Knerr, Patrick J.; Bindman, Noah A.; Velásquez, Juan E.; Donk, Wilfred A. van der

doi:10.1021/ja3017297

Cited by 92 publications

(138 citation statements)

References 47 publications

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“…Next, we explored the potential of leader-free macrocyclization following similar approaches used in other RiPP biosynthetic pathways 27,28 . We first attempted in trans cyclization of an ‘inactive’ substrate (MdnA Ac20–49 ) with the MdnA leader peptide MdnA 1–35 .…”

Section: Resultsmentioning

confidence: 99%

Structural basis for precursor protein–directed ribosomal peptide macrocyclization

Condurso

et al. 2016

Nat Chem Biol

109

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Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides whose members target proteases with potent reversible inhibition. The product structure is constructed by three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here, we describe the detailed structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases, MdnC and MdnB, interact with a conserved α-helix of the precursor peptide using a novel precursor peptide recognition mechanism. The results provide insight into the unique protein/protein interactions key to the chemistry, suggest an origin of the natural combinatorial synthesis of microviridin peptides and provide a framework for future engineering efforts to generate designed compounds.

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Section: Resultsmentioning

confidence: 99%

Structural basis for precursor protein–directed ribosomal peptide macrocyclization

Condurso

et al. 2016

Nat Chem Biol

109

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“…11,24–27 The decrease in processing efficiency can often be rescued in part by the addition of the leader peptide in trans . These studies have demonstrated that, in addition to providing substrate recognition, leader peptides activate their cognate biosynthetic machineries, presumably by biasing the enzymes to an active conformation.…”

Section: Resultsmentioning

confidence: 99%

Identification of an Auxiliary Leader Peptide-Binding Protein Required for Azoline Formation in Ribosomal Natural Products

et al. 2015

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Thiazole/oxazole-modified microcins (TOMMs) are a class of posttranslationally modified peptide natural products bearing azole and azoline heterocycles. The first step in heterocycle formation is carried out by a two component cyclodehydratase comprised of an E1 ubiquitin-activating and a YcaO superfamily member. Recent studies have demonstrated that the YcaO domain is responsible for cyclodehydration while the TOMM E1 homolog is responsible for peptide recognition during azoline formation. Although all characterized TOMM biosynthetic clusters contain this canonical TOMM E1 homolog (C domain), we also identified a second, highly divergent E1 superfamily member, annotated as an Ocin-ThiF-like protein (F protein), associated with more than 300 TOMM biosynthetic clusters. Here we describe the in vitro reconstitution of a novel TOMM cyclodehydratase from such a cluster and demonstrate that this auxiliary protein is required for cyclodehydration. Using a combination of biophysical techniques we demonstrate that the F protein, rather than the C domain, is responsible for engaging the peptide substrate. The C domain instead appears to serve as a scaffolding protein, bringing the catalytic YcaO domain and the peptide binding Ocin-ThiF-like protein into proximity. Our findings provide an updated biosynthetic framework that provides a foundation for the characterization and reconstitution of approximately 25 % of bioinformatically identifiable TOMM synthetases.

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“…In the LctM ConFusion system, the LctA leader peptide (spanning residues 1–24, violet) was fused to the N -terminus of the LctM enzyme via a (GlySer) 30 linker (red). 32 …”

Section: Figurementioning

confidence: 99%

“…Second, we recently engineered an LctM construct that is genetically fused to the LctA leader peptide in order to make a con stitutively active fusion enzyme (LctM ConFusion ) that catalyzes LctA dehydration without the need for exogenous leader peptide. 32 In addition, the dehydratase and cyclase activities of several LanM enzymes have been successfully split into two polypeptides, 25,33–35 an approach that we employ in this study in an attempt to decouple the LctM-catalyzed dehydration and cyclization reactions. Finally, we have recently developed mass spectrometry- and computation-based approaches that enable detailed kinetic and structural characterization of lanthipeptide biosynthetic pathways.…”

Section: Introductionmentioning

confidence: 99%

Leader Peptide Establishes Dehydration Order, Promotes Efficiency, and Ensures Fidelity During Lacticin 481 Biosynthesis

Thibodeaux

Wagoner

et al. 2016

J. Am. Chem. Soc.

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The mechanisms by which lanthipeptide synthetases control the order in which they catalyze multiple chemical processes are poorly understood. The lacticin 481 synthetase (LctM) cleaves eight chemical bonds and forms six new chemical bonds in a controlled and ordered process. Two general mechanisms have been suggested for the temporal and spatial control of these transformations. In the spatial positioning model, leader peptide binding promotes certain reactions by establishing the spatial orientation of the substrate peptide relative to the synthetase active sites. In the intermediate structure model, the LctM-catalyzed dehydration and cyclization reactions that occur in two distinct active sites orchestrate the overall process by imparting specific structure into the maturing peptide that facilitates the ensuing reaction. Using isotopically labeled LctA analogues with engineered lacticin 481 biosynthetic machinery and mass spectrometry analysis, we show here that the LctA leader peptide plays critical roles in establishing the modification order and enhancing the catalytic efficiency and fidelity of the synthetase. The data are most consistent with a mechanistic model for LctM where both spatial positioning and intermediate structure contribute to efficient biosynthesis.

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An Engineered Lantibiotic Synthetase That Does Not Require a Leader Peptide on Its Substrate

Cited by 92 publications

References 47 publications

Structural basis for precursor protein–directed ribosomal peptide macrocyclization

Structural basis for precursor protein–directed ribosomal peptide macrocyclization

Identification of an Auxiliary Leader Peptide-Binding Protein Required for Azoline Formation in Ribosomal Natural Products

Leader Peptide Establishes Dehydration Order, Promotes Efficiency, and Ensures Fidelity During Lacticin 481 Biosynthesis

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