Recombinant Tricyclic peptide MS-271

What is MS-271 and what is its structural composition?

MS-271 is a ribosomally synthesized and post-translationally modified peptide (RiPP) classified as a lasso peptide. It consists of 21 amino acid residues with a distinctive D-tryptophan at its C-terminus, which is unusual in ribosomally synthesized peptides. The lasso peptide features a characteristic N-terminal peptide chain-derived macrocyclic ring structure, which contributes to its remarkable stability and biological activity . The peptide derives from the precursor peptide MslA through several post-translational modifications including macrolactam formation and epimerization of the C-terminal tryptophan residue .

What organisms produce MS-271 naturally?

MS-271 is naturally produced by Streptomyces sp. M-271, a bacterial strain belonging to the actinomycetes family . Streptomyces species are gram-positive bacteria commonly found in soil and are well-known producers of bioactive secondary metabolites, including many clinically important antibiotics. The production of MS-271 by this specific strain involves a complex biosynthetic pathway encoded by a dedicated gene cluster that has been identified through draft genome sequencing .

What is the biosynthetic gene cluster of MS-271?

The MS-271 biosynthetic gene cluster (designated as msl) has been identified through draft genome sequencing of Streptomyces sp. M-271. The cluster contains several key genes encoding enzymes responsible for the post-translational modifications required for MS-271 biosynthesis, including:

• mslA: Encodes the precursor peptide containing all 21 amino acid residues

• mslC: Encodes a macrolactam synthetase

• mslB1: Encodes a precursor peptide recognition element

• mslB2: Encodes a cysteine protease

• mslE and mslF: Encode disulfide oxidoreductases

• mslH: Encodes a metallo-dependent peptide epimerase responsible for D-tryptophan formation

Heterologous expression studies in Streptomyces lividans have confirmed that this cluster contains all necessary genes for MS-271 production. Gene deletion experiments have further demonstrated that MslB1, MslB2, MslC, and MslH are indispensable for MS-271 biosynthesis .

How is D-tryptophan introduced in the structure of MS-271?

The D-tryptophan at the C-terminus of MS-271 is introduced through an epimerization process catalyzed by MslH, a dedicated epimerase encoded within the MS-271 biosynthetic gene cluster. Unlike many other D-amino acid-containing peptides where D-amino acids are incorporated directly or generated through non-ribosomal peptide synthetase systems, MS-271 undergoes post-translational epimerization after ribosomal synthesis of the precursor peptide .

MslH catalyzes the epimerization at the Cα center of the C-terminal Trp21 of the precursor peptide MslA, converting it to epi-MslA. Structural and biochemical studies have revealed that MslH is a metal-dependent epimerase with a calcineurin-like fold, employing acid/base chemistry to facilitate the reversible epimerization of the terminal tryptophan residue .

What is the mechanism of action of the MslH epimerase in MS-271 biosynthesis?

MslH employs a sophisticated catalytic mechanism to perform the epimerization of the C-terminal tryptophan in MS-271 biosynthesis. Crystal structure analysis has revealed that MslH is a metallo-dependent peptide epimerase with a calcineurin-like fold. The enzyme utilizes two pairs of His/Asp catalytic residues that are electrostatically tethered to a six-coordination motif containing a Ca(II) ion via water molecules .

The reaction mechanism involves acid/base chemistry to facilitate the reversible epimerization of the C-terminal Trp21. Specifically:

Site-directed mutagenesis studies, docking simulations, and ICP-MS analyses have confirmed the essential role of these residues and the metal cofactor in the epimerization activity of MslH .

How can experimental design approaches be optimized for studying MS-271 structure-function relationships?

To effectively study MS-271 structure-function relationships, researchers should implement Design of Experiments (DoE) and Response Surface Methodology (RSM) approaches. These statistical techniques allow for systematic exploration of multiple variables simultaneously while minimizing the number of experiments required.

The general workflow should include:

• Screening Phase: Identify significant factors affecting MS-271 production or activity using fractional factorial designs (e.g., 2^k-p designs)

• Optimization Phase: Use RSM to find optimal conditions for MS-271 synthesis or activity

• Validation Phase: Confirm the predicted optimal conditions experimentally

When setting experimental bounds, all factors should be normalized to a range between -1 and +1 to allow proper statistical analysis, as shown in the following table :

Effects can be calculated using the formula:
Effect = (ȳ₊ - ȳ₋)/divisor

Where ȳ₊ is the average response at the high level and ȳ₋ is the average response at the low level of each factor .

How do metal ions influence the epimerization activity of MslH?

Metal ions play a crucial role in the epimerization activity of MslH. Crystallographic studies combined with ICP-MS analysis have demonstrated that MslH is a metallo-dependent epimerase that utilizes Ca(II) ions for its catalytic activity .

The Ca(II) ion in MslH:

• Forms a six-coordination motif with protein residues and water molecules

• Positions the catalytic His/Asp residues optimally for proton abstraction and donation

• Stabilizes the reaction intermediates during the epimerization process

• Creates an electrostatic environment conducive to the acid/base chemistry required for epimerization

Researchers investigating MslH activity should consider:

• Testing various metal ions (Ca²⁺, Mg²⁺, Mn²⁺, Zn²⁺) to determine specificity

• Using metal chelators (EDTA, EGTA) to confirm metal dependency

• Performing site-directed mutagenesis of metal-coordinating residues

• Monitoring metal binding through spectroscopic methods (ICP-MS, circular dichroism)

What methodologies are recommended for purification and characterization of recombinant MS-271?

Purification and characterization of recombinant MS-271 requires a systematic approach integrating multiple techniques. Based on successful recombinant protein production strategies, the following workflow is recommended:

Production System Selection:

• E. coli expression systems can be suitable hosts, though optimization of strain selection is crucial

• Consider strains like E. coli DH5α which have shown success with other recombinant peptides

• Maintain high antibiotic concentrations (e.g., 1,000 μg/ml ampicillin) to ensure plasmid retention

Purification Protocol:

• Cell lysis using sonication or pressure homogenization

• Initial separation by ammonium sulfate precipitation

• Ion exchange chromatography

• Hydrophobic interaction chromatography

• Preparative electrophoresis for final purification (yields approximately 6%)

Characterization Methods:

• HPLC analysis with UV detection

• Mass spectrometry for exact mass determination

• Circular dichroism to confirm secondary structure

• NMR spectroscopy for detailed structural analysis

• Immunological assays to confirm antigenicity

How can site-directed mutagenesis be utilized to explore the catalytic mechanism of MslH?

Site-directed mutagenesis provides a powerful approach to dissect the catalytic mechanism of MslH. Based on crystallographic studies revealing the key catalytic residues and metal-binding sites of MslH, the following mutagenesis strategy is recommended: