Expansion of ribosomally produced natural products: a nitrile hydratase- and Nif11-related precursor family

Abstract

Background: A new family of natural products has been described in which cysteine, serine and threonine from ribosomally-produced peptides are converted to thiazoles, oxazoles and methyloxazoles, respectively. These metabolites and their biosynthetic gene clusters are now referred to as thiazole/oxazole-modified microcins (TOMM). As exemplified by microcin B17 and streptolysin S, TOMM precursors contain an N-terminal leader sequence and C-terminal core peptide. The leader sequence contains binding sites for the posttranslational modifying enzymes which subsequently act upon the core peptide. TOMM peptides are small and highly variable, frequently missed by gene-finders and occasionally situated far from the thiazole/oxazole forming genes. Thus, locating a substrate for a particular TOMM pathway can be a challenging endeavor.

Results: Examination of candidate TOMM precursors has revealed a subclass with an uncharacteristically long leader sequence closely related to the enzyme nitrile hydratase. Members of this nitrile hydratase leader peptide (NHLP) family lack the metal-binding residues required for catalysis. Instead, NHLP sequences display the classic Gly-Gly cleavage motif and have C-terminal regions rich in heterocyclizable residues. The NHLP family exhibits a correlated species distribution and local clustering with an ABC transport system. This study also provides evidence that a separate family, annotated as Nif11 nitrogen-fixing proteins, can serve as natural product precursors (N11P), but not always of the TOMM variety. Indeed, a number of cyanobacterial genomes show extensive N11P paralogous expansion, such as Nostoc, Prochlorococcus and Cyanothece, which replace the TOMM cluster with lanthionine biosynthetic machinery.

Conclusions: This study has united numerous TOMM gene clusters with their cognate substrates. These results suggest that two large protein families, the nitrile hydratases and Nif11, have been retailored for secondary metabolism. Precursors for TOMMs and lanthionine-containing peptides derived from larger proteins to which other functions are attributed, may be widespread. The functions of these natural products have yet to be elucidated, but it is probable that some will display valuable industrial or medical activities.

Figure 1

The biosynthesis and defining chemical features of TOMM and lanthionine-containing natural products. (A) Through the action of a trimeric 'BCD' complex, consisting of a cyclodehydratase (green), dehydrogenase (yellow) and docking/scaffolding protein (blue), thiazoles and (methyl/oxazoles are incorporated onto a peptidic scaffold (black). These heterocycles are synthesized from serine/threonine (X = O) and cysteine (X = S) residues of an inactive precursor peptide and yield a bioactive natural product. The chemical transformations carried out by the cyclodehydratase the dehydrogenase are shown, along with the corresponding mass change in Daltons. (B) A bifunctional synthase, LanM, catalyzes both the dehydration and Michael-type addition steps required to synthesize lanthionine crosslinks.

Figure 2

The genetic organization of TOMM and lanthionine biosynthetic clusters that utilize NHase- and Nif11-related precursor peptides. Genomic regions are shown from selected organisms in which the precursor peptides are clustered with the cognate modification enzymes. In most cases, a transport system is also visible in the local region. The TOMM precursors represented by Burkholeria cenocepacia (dark gray ORFs, NHLP-Burk) are accompanied by a large, Ser/Thr kinase. Highly similar clusters have been identified in Acidovorax avenae and Delftia acidovorans. In several species, precursors shown in black (NHLP) and light grey [Nif11-derived peptide (N11P)] may cluster with each other as well as with other modification and transporter genes. Note: only those precursors closest to the cyclodehydratase-docking scaffold protein or LanM-like lanthionine synthase are shown. For instance, Pelotomaculum thermopropionicum has 12 NHLPs (only two are shown) and seven N11P family precursors, while Cyanothece sp. PCC 7425 and P. marinus have 18 and 29 predicted N11P precursors, respectively (eight and seven are shown). Transport proteins, including those homologous to HlyD (type I secretion, purple) and ABC transporters (red), correspond to the transport genes detected by PPP.

Figure 3

Alignment of nitrile hydratase (NHase) with nitrile hydratase leader peptide (NHLP) sequences. (A) Fourteen members of the NHase alpha subunit protein family (TIGR01323), identified by locus tags, are shown aligned to the leader sequences of 28 members of the NHLP family (TIGR03793). Along the top of the figure is a colour-coded region depicting the anticipated secondary structure for that region (red, alpha-helix; blue, loop; green, beta-sheet). Relative to NHase, the NHLP sequences exhibit a 63-residue deletion that carries the residues required for iron/cobalt ligation, the CxxCSC motif. Without the ability to bind the required catalytic metal, the truncation seen in NHLP is presumed to abolish NHase activity. Shown in the truncated region is the canonical metal coordination architecture, with two of the three Cys thiol ligands being oxidized to sulphenic and sulphinic acids. Also shown is the putative leader sequence cleavage site for NHLP, which is not conserved with full-length NHase. (B) Crystal structure of the NHase from Bacillus smithii, the most closely related NHase to the NHLP family with a known structure [38]. The N- and C-termini have been labelled, the metal centre is shown as a cyan sphere and the beta subunit has been omitted for clarity. The colour coding is by secondary structure as in panel A with the addition of the N- and C-terminal extensions (grey) that are not included in the alignment. The final residue of the NHase alignment, Arg, is shown in blue stick format. (C) Same as in panel B, but with the insertion-deletion region omitted in NHLP's shown in wheat. This figure was generated using a previously described, web-based program [36] and PyMOL.

Figure 4

Sequence alignment of nitrile hydratase leader peptides (NHLPs) from natural combinatorial biosynthetic clusters. ClustalW alignment [44] of NHLPs from a putative thiazole/oxazole-modified microcin cluster from (A)Pelotomaculum thermopropionicum SI (12 sequences) and (B) Azospirillum sp. B510 (eight sequences). Possible sites for thiazole formation are highlighted in cyan and sites of potential oxazole and methyloxazole formation are yellow. The locus tag is given to the left of the sequence and the amino acid position is given on the right. An asterisk implies an invariant residue, while the semicolon and period show positions that are highly and moderately related, respectively. Underlined red text indicates the putative leader peptide cleavage motif.

Figure 5

Sequence alignment of nitrile hydratase leader peptides (NHLPs) from Nostdoc punctiforme PCC 73102. Shown is a ClustalW alignment of six selected NHLP substrates. N. punctiforme PCC 73102 has at least 16 total substrates, half of which are NHLP and the other half N11P. The coloring scheme and notation are identical to Figure 4.

Figure 6

Sequence logo comparison of classical Gram-positive bacteriocin and nitrile hydratase leader peptides (NHLP)/Nif11-derived peptides (N11Ps). (A) Logo depiction of TIGR01847, which is representative of the sequence found near the peptide cleavage site of Gram-positive bacteriocins. (B) Logo for the NHLP and N11P families taken together. In addition to the classic Gly-Gly cleavage motif (positions 12-13), the two logos show an abundance of acidic residues at positions 1, 4 and 6 and a significant preference for Leu at position 2 and 7, Ser/Thr at position 3, and either Val/Leu at position 10. This figure was generated online using a published algorithm [53].