A Most Versatile Enzyme

Many enzymes have the flexibility to operate on a pool of similarly shaped substrates, but rarely is a single enzyme so versatile that it can catalyze a reaction on substrates with radically different shapes.

Now, in the Prochlorococcus genus of ocean-dwelling cyanobacteria, researchers have discovered and begun to characterize an enzyme that can loop together either a serine or a threonine with a cysteine, regardless of where those amino acids are found on peptide chains of up to 32 residues long. The final ringed products are so-called lanthionine-containing peptides, a family of natural products used as antibiotics or scaffolds for them.

"This enzyme's promiscuity is unbelievable," says Wilfred A. van der Donk, a chemist at the University of Illinois, Urbana-Champaign, who with Sallie W. Chisholm of Massachusetts Institute of Technology led the team of researchers who studied the ability of the so-called ProcM enzyme to catalyze formation of 29 structurally unique cyclic peptides (Proc. Natl. Acad. Sci. USA, DOI: 10.1073/pnas.0913677107).

Although enzymes that catalyze similar cyclizations had already been found, most previously known enzymes operate only on a single substrate. "ProcM can cyclize rings of drastically different sizes, a ring within a ring, overlapping rings, rings that form in the direction of a peptide's carboxyl to amino termini, and vice versa," van der Donk adds.

The enzyme's promiscuity is only rivaled by "the relaxed specificity of P450 enzymes, which decorate terpenoid products in plants," but unlike ProcM, P450 enzymes get help from plant cyclases to form ring structures, van der Donk says.

ProcM operates on peptides that are encoded by genes, produced by the ribosome, and have two distinct components: First there's a conserved sequence of some 30 amino acids that help ProcM recognize the peptide but eventually get cleaved off. Then there's an extremely variable selection of 13 to 32 residues that ProcM can loop together into a lanthionine-containing peptide.

The cyclization happens in two sequential steps. The first is a dehydration reaction in which a hydroxyl group is removed from a threonine or serine. The second step cyclizes the peptide by linking the dehydroxylated side chain with the thiol group of a cysteine, which could be next door or dozens of residues away.

"It's hard to imagine an active site that can accept this range of substrates," van der Donk says. The team is currently trying to solve the enzyme's crystal structure to figure out how it can be so versatile. The structure may help protein engineers figure out what makes ProcM so multitalented and apply the enzyme's skills for applications as varied as building new antibiotic leads and basic chemical biology.

The enzyme was found in Prochlorococcus, which are the smallest and most abundant photosynthetic cells in the oceans and also have a tiny genome. A "particularly significant" aspect of this study, says Bradley Moore, who studies marine natural products at the University of California, San Diego, is that even microbes with small genomes have the capacity to synthesize secondary metabolites that may be crucial for their specialized lifestyles. "Previously, the consensus was that only microbes with large genomes had the luxury of numerous secondary metabolic pathways"—such as polyketide synthase (PKS) or nonribosomal peptide synthase (NRPS) assembly machinery, which require genomes three to four times larger than Prochlorococcus—to make specialized products, Moore adds.

"Prochlorococcus lives in the nutrient-poor oceans, where most free-living bacteria can't afford to replicate the huge genomes for PKS or NRPS machinery, so it makes simple and efficient use of the ribosome and ProcM to produce complex metabolites."