Lasso peptides are a class of ribosomally-synthesized and posttranslationally-modified natural products with diverse bioactivities. This review describes the structure and function of all known lasso peptides (as of mid-2012) and covers our current knowledge about the biosynthesis of those molecules. The isolation and characterization of lasso peptides are also covered as are bioinformatics strategies for the discovery of new lasso peptides from genomic sequence data. Several studies on the engineering of new or improved function into lasso peptides are highlighted, and unanswered questions in the field are also described.
Microcin J25 (MccJ25) is a ribosomally synthesized antimicrobial peptide that has an unusual threaded lasso structure in which the C-terminal "tail" of the peptide is fed through a macrocyclic "ring" formed by the N-terminal residues. Production of MccJ25 in Escherichia coli is dependent upon a four-gene cluster encoding the structural gene mcjA, two maturation enzymes mcjB and mcjC, and an immunity factor, mcjD, in the form of an MccJ25 export pump. Here we have developed a system for orthogonal control of the expression of mcjA and mcjD, thus permitting independent control of MccJ25 production and export/immunity in E. coli. We used this system to screen saturation mutagenesis libraries targeted to either the ring or tail portions of MccJ25 and discovered nearly 100 new MccJ25 variants that retain antimicrobial function. While multiple amino acid substitutions in the tail portion of the peptide are well-tolerated, mutagenesis of the ring portion of the peptide is detrimental to the antimicrobial function of MccJ25. We demonstrated that the decreased function of the ring variants is due to the inability of these variants to be transported to the cytoplasm of susceptible strains. Additionally, we found several MccJ25 variants from the tail library with improved efficacy toward the MccJ25-sensitive strains E. coli and Salmonella enterica serovar Newport with the best variants exhibiting a nearly 5-fold increase in potency. The results described here provide further evidence that diverse amino acid sequences can be tolerated by the rigid lasso peptide fold.
The conserved threonine (Thr) residue in the penultimate position of the leader peptide of lasso peptides microcin J25 and capistruin can be effectively replaced by several amino acids close in size and shape to Thr. These findings suggest a model for lasso peptide biosynthesis in which the Thr sidechain is a recognition element for the lasso peptide maturation machinery.
The antimicrobial peptide microcin J25 (MccJ25) is matured by two enzymes, McjB and McjC, from a 58 amino acid (aa) preprotein, McjA, into its final 21 aa lasso topology. Herein we have investigated the role of the leader peptide of McjA and found that only the eight C-terminal amino acids of this leader peptide are required for maturation of MccJ25. There is a high content of lysine residues in the McjA leader peptide, but herein we also demonstrate that these charged amino acids do not play a major role in the maturation of MccJ25. Alanine scanning mutagenesis studies revealed that the Thr-35 residue in the leader peptide is critical for correct processing of McjA into mature MccJ25. In the absence of detailed structural and biochemical data about McjB and McjC, these studies allow us to propose a putative role for the leader peptide as a simple motif for docking of the McjA preprotein in the maturation enzymes.
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