RESEARCH

Numerous reports of multi-drug resistant bacterial strains have appeared in recent years. Several bacterial strains may soon become immune to all commercially available antibiotics. It is evident that without a renewed focus on antibiotic research, we risk of returning to an era in which common infectious diseases are untreatable and deadly. The van der Donk group focuses on the discovery, mode of action, and mechanism of biosynthesis of two classes of antibiotics that are underexplored and have great potential for human therapeutic use: ribosomally synthesized and post-translationally modified peptides (RiPPs) and phosphonate antibiotics. Tremendous advances in DNA sequencing are providing exciting new opportunities in natural product discovery through genome mining, and the unique biosynthetic pathways of RiPPs are well-suited for synthetic biology approaches.

Lantibiotics, Glycocins, and Thiopeptides

Genome sequencing efforts have demonstrated that RiPPs are a large and diverse family of natural products. The van der Donk lab is focused on understanding the biosynthesis and biochemical function of these compounds, in particular three of the many classes:

  • Lantibiotics are lanthipeptide antibacterial agents, thus named for the presence of lanthionine thioether crosslinks. These peptides are post-translationally modified in a multi-step process. Serine and threonine residues are dehydrated to give dehydroalanine and dehydrobutyrine residues, respectively. A cyclase then catalyzes the regio- and stereoselective conjugate additions of cysteine residues onto these dehydro amino acids.
  • Glycocins are antimicrobial peptides that often contain an unusual glycosylated Cys residue.
  • Thiopeptides typically contain several different modifications and are made through a series of unusual enzymatic reactions.

Mode of action

In order to clarify how lantibiotic-producing bacteria achieve immunity against their own compounds, studies have focused on the interaction between lantibiotics and their molecular target, lipid II. The lab has also expanded into the study of the glycocin sublancin, which has a different target and mode of action. Promising forays into this area include structural characterization of sublancin by NMR and genetic studies of sublancin-resistant mutants.

Discovery

Analysis of available genome sequences has revealed many RiPP gene clusters, some of which contain enzymes not usually associated with RiPP biosynthesis or peptide sequences that have not been characterized. Ongoing efforts in the laboratory are focused on the structural and functional characterization of these novel RiPPs and the enzymes that produce them. These studies have revealed previously unknown post-translational modifications.

Bioengineering

Improved understanding of lanthipeptide biosynthesis has permitted bioengineering of both the mature lantibiotics and their synthases. The successful incorporation of non-proteinogenic amino acids into various lantibiotics has enabled new approaches for leader peptide removal and chemical labeling. In this vein, the lab is interested in expanding the bioactivities and affinities of lanthipeptides by developing selection platforms for lanthipeptide variants. In tandem to these peptide-focused approaches, the enzymes involved in lanthipeptide biosynthesis have been successfully employed to process substrate variants or non-endogenous substrates.

Mechanism

Key to these bioengineering efforts is a mechanistic understanding of the enzymes involved in RiPP biosynthesis. Studies in the lab have provided kinetic insight into these multi-step reactions and quantification of the binding between lanthipeptide synthases and substrates. Additionally, the lab has elucidated the elusive mechanism of the LanB enzymes that dehydrate class I lantibiotics, such as the food preservative nisin. These studies have enabled the in vitro reconstitution of LanB-like enzymes involved in the biosynthesis of the antimicrobial thiomuracin.

Phosphonates

Phosphonate compounds are relatively unexplored with respect to biosynthesis, despite their potential use in antibacterial, antiviral, and antiparasitic therapies. Moreover, phosphonates are used extensively in agriculture as herbicides and pesticides. Phosphonates and phosphinates mainly derive from phosphoenolpyruvate and are effective antibacterial and antifungal agents because the P-C bond is resistant to hydrolysis. Many exert their biological action as mimics of carboxylic acids or phosphate esters. Compounds in this class include the highly effective anti-malarial agents FR900098 and fosmidomycin; fosfomycin, an FDA approved drug for the treatment of acute cystitis; and phosphinothricin, a potent herbicide. Various formulations of phosphinothricin are widely used in agriculture, with annual sales in excess of $200 million per year.

The van der Donk group is part of a multidisciplinary and multi-laboratory effort to both better understand the biosynthetic pathways of phosphonate natural products and also to discover new phosphonate compounds. Our contribution to this effort involves investigation of biosynthetic enzymes, organic synthesis of phosphonate antibiotics, and spectroscopic studies on newly discovered natural products. This program is a collaboration with the laboratories of Bill Metcalf (Microbiology), Huimin Zhao (ChBE), and Satish Nair (Biochemistry).

Special Acknowledgement to the contribution of Dr. M. C. Walker, Dr. L. Repka, Dr. M. A. Funk, Ms. E. Ulrich, Ms. X. Zhao for the content of this page.

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