Tuesday, February 8, 2011

The dark side of amino acyl tRNA synthetases - The case of cyclodipeptide synthases

It is textbook knowledge that amino acyl tRNA synthetases (AAtRS) catalyze the esterification of an amino acid with its cognate tRNA. The charged tRNAs then incorporates the cognate amino acid into a growing polypetide during translation. However, increasing evidence from recent studies point to non-ribosomal roles for these ancient enzymes and charged tRNAs, such as in peptide metabolite biosynthesis and cell wall biosynthesis. Recently, we reported a few such distinct families and contexts, one of which is described below.

Cyclodipeptides are fascinating molecules formed by the condensation of two amino acids resulting in the formation of a diketopiperazine. Natural cyclopeptides have diverse roles as antibacterials, antifungals, antitumor and immuno-suppressive agents. Most cyclopeptides are synthesized by giant multi-domain proteins called non-ribosomal peptide synthetases. However, in an exciting recent study, a new family of enzymes that catalyze cyclopeptide formation was reported. The study showed that a distinct enzyme AlbC catalyzes the formation of the cyclodipeptide Albonoursin. AlbC was unusual in that it required charged amino acids in the form of aminoacylated tRNAs as substrates, thus defining a hitherto unreported enzyme family. The same study also reported other related enzymes involved in the synthesis of the cyclodipeptides pulcherriminic acid ( a red pigment) in Bacillus subtilis and cYY (cyclodityrosine) in Mycobacterium tuberculosis. This novel enzymatic family was called the cyclodipeptide synthase (CDPS). Click here to go to the article.

Using, sensitive sequence analysis methods, we showed that the AlbC -like CDPSs are related to Class I aminoacyl tRNA synthetases such as tyrosine tRNA synthetase and tryptophan tRNA synthetase. The CDPSs however lack the key amino acids required for ATPase activity (called the HIGH motifs) normally seen in Class I AAtRS. They instead contain a distinct constellation of residues that we predict are required to form an amide linkage ( as in cyclodipeptides) from aminoacylated tRNAs, as opposed to the adenylation followed by ester formation seen in ancestral AAtRS (See figure below). Thus these enzymes bind aminoacylated tRNAs like classical AAtRSs but catalyze a completely different reaction. The crystal structure of the Mycobacterium tuberculosis cyclodityrosine synthetase, published subsequent to our publication, confirmed our prediction. You can access this study here and the New and Views about these discoveries.

Sequence searches with CDPSs revealed them to be present in a wide phyletic range of free-living bacteria, and also in intracellular parasitic bacteria such as Legionella, Rickettsiella and certain chlamydiae. In the latter organisms, the products of the CDPSs may be involved in the survival in or manipulation of host cells. We also detected CDPSs in pathogenic fungi (where it may have a bioactive effect on its host) and in the annelid Platynereis, where the CDPS is induced as part of an antibacterial innate immune response. Thus cyclodipeptides might play a role as endogenously encoded antibiotics in the immune response of certain animals.

Contextual analysis suggests that these enzymes are found in the neighborhood of cytochrome P450s, nitroreductases, 2OGFeDO-like hydroxylases, and other peptide ligases that might further modify the cyclodipeptide (some of this has been shown experimentally). Yet another fascinating context, seen in Actinosynnema mirum and Streptomyces sp AA4, is the presence of CDPSs in the neighborhood of enzymes that suggests that it might also use aminoacyl-coAs as substrates. Get ready for a whole new world of cyclodipeptides in the coming years. You can access our article here. Feel free to peruse the extensive supplementary material and watch this space for more.