A Unique TryptophanC-Prenyltransferase from the Kawaguchipeptin Biosynthetic Pathway

Abstract

Cyanobactins are are rapidly growing family of linear and cyclic peptides produced by cyanobacteria. Kawaguchipeptins A and B, two macrocyclic undecapeptides reported earlier from Microcystis aeruginosa NIES-88, are shown to be products of the cyanobactin biosynthetic pathway. The 9 kb kawaguchipeptin (kgp) gene cluster was identified in a 5.26 Mb draft genome of Microcystis aeruginosa NIES-88. We verified that this gene cluster is responsible for the production of the kawaguchipeptins through heterologous expression of the kgp gene cluster in Escherichia coli. The KgpF prenyltransferase was overexpressed and was shown to prenylate C-3 of Trp residues in both linear and cyclic peptides in vitro. Our findings serve to further enhance the structural diversity of cyanobactins to include tryptophan-prenylated cyclic peptides.

Cyanobactins are a family of ribosomally-synthesized and posttranslationally modified peptides (RiPPs) produced by cyanobacteria.[1–6] Cyanobactin posttranslational modifications include N-to-C macrocyclization; epimerization; heterocyclization to form thiazolines and oxazolines; oxidation of heterocycles to thiazoles and oxazoles; N-methylation of His; O-prenylation on Ser, Thr, and Tyr; and N-prenylation.[2, 7, 8] Biological activities that have been reported for cyanobactins include anticancer, antimalarial, antibacterial, and protease inhibitory activity.[2]

The posttranslational prenylation of cyanobactins is catalyzed by a prenyltransferase enzyme encoded within the cyanobactin biosynthetic gene cluster, which uses 3-methyl-but-2-en-1-yl group derived from dimethylallyl pyrophosphate (DMAPP).[9–12] Although a putative prenyltransferase gene is present in all known cyanobactin gene clusters, only a few of the cyanobactins are known to be prenylated, including prenylagaramides, aestuaramides, trunkamides, and anacyclamides.[4, 6, 10, 12] The known cyanobactin prenyltransferases are O-prenyltransferases that catalyze the O-prenylation of Tyr, Thr, and Ser in forward or reverse orientation. C-prenylated cyanobactins have been shown to be synthesized originally as O-prenylated peptides that later undergo a Claisen rearrangement to yield C-prenylated peptides.[12]

Kawaguchipeptins are macrocyclic undecapeptides produced by the cyanobacterial strain Microcystis aeruginosa NIES-88.[13, 14] Two variants of kawaguchipeptin have been reported (Scheme 1). Kawaguchipeptin A contains two C-3-prenylated tryptophan residues and a d-Leu residue.[13] Kawaguchipeptin B consists of solely unmodified amino acids and is reported to show antimicrobial activity against Staphylococcus aureus.[14] Herein, we report a genome sequence for Microcystis aeruginosa NIES-88, identify the kawaguchipeptin biosynthetic gene cluster, and confirm enzymatic prenylation activity.

Scheme 1.

The cyclic undecapeptides Kawaguchipeptin A (1) and kawaguchipeptin B (2) reported from Microcystis aeruginosa NIES-88. Kawaguchipeptin A contains two C-3 prenylated tryptophans (highlighted).

The cyclic undecapeptides kawaguchipeptin A (1) and kawaguchipeptin B (2) reported from Microcystis aeruginosa NIES-88. Kawaguchipeptin A contains two C-3-prenylated tryptophan residues (highlighted). Prenylation of C-3 is coupled to the formation of a new ring formation as a result of bond formation between C-2 and NH in the main chain.

We obtained a 5.26 Mb genome sequence for Microcystis aeruginosa NIES-88 by using a combination of 3 kb 454 sequencing and short-insert illumina Miseq paired-end data, which were subsequently assembled into 29 scaffolds. A total of 4,996 genes, including 4 ribosomal RNA operons and 41 tRNAs, were annotated from this genome. The kawaguchipeptin precursor gene was identified through tBLASTn using the predicted kawaguchipeptin peptide backbone (WLNGDNNWSTP). The KgpE precursor peptide was found to encode three exact copies of the WLNGDNNWSTP core (Figure 1). The KgpE precursor peptide was encoded in a 9 kb gene cluster (kgp) together with the KgpA and KgpG cyanobactin proteases, as well as the putative KgpF prenyltransferase (Figure 1). The KgpF prenyltransferase shows just 20–45% identity to known and putative cyanobactin prenyltransferases. A homologue of cyclodehydrase (PatD) was not detected in the gene cluster, which is consistent with the absence of heterocyclized amino acids in the cyclic peptides.

Figure 1.

The kawaguchipeptin biosynthetic pathway in Microcystis aeruginosa NIES-88. (A) The 10 kb biosynthetic gene cluster that consists of six biosynthetic genes (indicated by grey and black) are organized in a single operon and have homology to genes present in other known cyanobactin gene clusters. The open arrows indicate the genes that encode proteins with unknown functions. The kgpE precursor gene (black) encodes the 87 amino-acid precursor protein (B). Three identical copies of the undecapeptide core are encoded by the single precursor gene (highlighted in gray).

The kawaguchipeptin biosynthetic pathway in Microcystis aeruginosa NIES-88.[23] A) The 9 kb kgp biosynthetic gene cluster consists of six biosynthetic genes (shown in blue), organized in a single operon, that show homology to genes present in other known cyanobactin gene clusters. The grey arrows indicate genes that encode proteins with unknown functions. B) The kgpE precursor gene encodes the 87 amino acid precursor peptide, which contains three identical copies of the undecapeptide core (highlighted in grey).

To demonstrate that the kgpA-G genes encode kawaguchipeptin production in vivo, we cloned the entire kgp operon into a broad host range yeast/bacteria shuttle vector pMQ123i[15] and placed kgpA downstream of a pTac promoter to generate the expression plasmid pDK-kgp1 (Figure S1 in the Supporting Information). This construct allowed the regulated expression of the kgpA-G genes in E. coli TOP10. Liquid chromatography with high-resolution mass spectrometry (LC–HRMS) guided metabolite profiling of E. coli cells transformed with pDK-kgp1 revealed the presence of 2, for which the LC retention time and HRMS profile matched authentic 2 isolated from M. aeruginosa NIES-88 (Figure 2, traces 2–3, and Figure S2). E. coli cells carrying pDK-kgp1 alone, however, did not produce 1 (Figure 2, trace 5), which is potentially derived from 2 by bisprenylation at C-3 of Trp by prenyltransferase KgpF. We hypothesized that this observation may have been due to the lack of sufficient endogenous prenyl donor dimethylallyl pyrophosphate (DMAPP) in E. coli. To overcome this problem, we co-transformed E. coli with pDK-kgp1 and the plasmid pMBI,[16] which harbors four yeast mevalonate-dependent isoprenoid pathway biosynthetic genes that can convert mevalonate to isopentenyl pyrophosphate (IPP), a precursor to DMAPP. Co-expression of the pDK-kgp1 and pMBI genes in E. coli TOP10 supplied with exogenous mevalonolactone 3 (1.0 mm) led to the production of both 1 and 2 that matched their authentic standards from M. aeruginosa NIES-88 (Figure 2, traces 1–2, 6–7, and Figure S3), as assessed by LC–HRMS. These experiments establish that the kgpA-G genes confer 1 and 2 biogenesis in vivo. In addition, the coexistence of 1 and 2 in E. coli cells transformed with both pDK-kgp1 and pMBI and the lack of 1 in E. coli cells transformed with pDK-kgp1 alone strongly suggest that 2 is the direct biosynthetic precursor to 1 and bis-prenylation by KgpF is likely the last enzymatic step in the biosynthetic maturation of kawaguchipeptins.

Figure 2.

Heterologous expression of the kgp operon in E. coli demonstrates that the kgpA-G genes confer the production of 1 and 2 in vivo. Extracted ion chromatographs of LC–HRMS analysis of: Authentic 1 and 2 from M. aeruginosa NIES-88 (1, 2); 2 in E. coli transformed with pDK-kgp1 (3); 1 in E. coli transformed with pDK-kgp1 (4); 1 in E. coli co-transformed with pDK-kgp1 and pMBI and supplied with mevalonolactone 3 (5); and 2 in E. coli co-transformed with pDK-kgp1 and pMBI and supplied with mevalonolactone 3 (6).

Heterologous expression of the kgp operon in E. coli demonstrates that the kgpA-G genes confer the production of 1 and 2 in vivo. Extracted ion chromatographs of LC–HRMS analysis of: Authentic 1 and 2 from M. aeruginosa NIES-88 (1, 2); 2 in E. coli transformed with pDK-kgp1 (3); 1 in E. coli transformed with pDK-kgp1 (4); 1 in E. coli co-transformed with pDK-kgp1 and pMBI and supplied with mevalonolactone 3 (5); and 2 in E. coli co-transformed with pDK-kgp1 and pMBI and supplied with mevalonolactone 3 (6).

We overexpressed and purified the recombinant KgpF from E. coli and assessed the enzymes ability to process a range of cyclic and linear peptides and to use isopentenyl pyrophosphate (IPP), DMAPP, and geranyl pyrophosphate (GPP; Table 1 and Figures S4–S24).

Table 1.

Substrates and co-factors used for the in vitro reactions.

Substrate	Co-factor	Mono-prenylation
Cyclic [WLNGDNNWSTP] (2)	DMAPP	+
Cyclic [WLNGDNNWSTP] (2)	IPP	+
Cyclic [WLNGDNNWSTP] (2)	GPP	-
Cyclic [TSQIWGSPVP] (3)	DMAPP	+
Cyclic [SAQWQNFGVP] (4)	DMAPP	+
Cyclic [HAFIGYDQDPTGKYP] (5)	DMAPP	+
Cyclic [RERFVYP] (6)	DMAPP
Cyclic [LIGIMHP] (7)	DMAPP
WLNGDNNWSTP (8)	DMAPP	+
WLNGDNNWSTPAYDG (9)	DMAPP	+
EDWYFDHPAYDG (10)	DMAPP	-
VPWPFPAYDG (11)	DMAPP	-
Boc-Trp (12)	DMAPP	-
Boc-Trp (13)	IPP	-
Boc-Tyr (14)	DMAPP	-
Boc-Tyr (15)	IPP	-

[+] Product detected, [-] Product not detected.

Our results show that the enzyme processes a second Trp residue within macrocyclic peptide substrate 2, whereas in the linear peptide 9, despite 40 h of incubation, only a single modification was observed with DMAPP as the cofactor (Table 1; Figures S5–S7, S18–S20). The catalytic activity of the enzyme decreased when IPP was used as a cofactor instead of DMAPP, as seen in the processing of one Trp residue out of two in kawaguchipeptin B (2) when IPP instead of DMAPP was used (Table 1; Figures S8). We did not detect any processing of other residues in the linear or macrocyclic peptide substrates. The enzyme did not process Boc-Trp (13) in presence of DMAPP or IPP. Interestingly, the enzyme could not use GPP as a cofactor. According to these results, the selectivity of prenylation in linear substrates can be explained by the necessity of the Trp residue to be sandwiched between two residues. Terminal Trp residues would presumably be too strongly solvated to bind efficiently the enzymatic site and undergo transformation (Figures S20, S22).

O- or C-prenylation of Tyr, Ser, and Thr in forward or reverse orientation have been observed for cyclic cyanobactins.[2, 5, 12] C-prenylated peptides like the C-prenylated Tyr in aestuaramides have been reported.[12] However, the latter is the result of reverse O-prenylation on the oxygen atom of Tyr followed by Claisen rearrangement.[12] The biochemical characterization of C-3 Trp prenylation[13] and demonstration of the presence of a homologue of the prenyltransferase gene kgpF in the gene cluster confirm that this is a direct posttranslational modification, which is rare in cyanobactins. To our knowledge, C-3 prenylation of Trp through posttranslational modification of a peptide has been demonstrated only once before for the ComX peptide, a pheromone produced by Bacillus subtilis and related bacilli.[17, 18]

Trp prenylation is common in some plants and bacteria, but mostly in fungi, and the respective prenyltransferases catalyze the addition of a dimethylallyl group to Trp during the synthesis of secondary metabolites.[19–21] The synthesis and biosynthesis of these compounds, particularly indole alkaloids that contain prenylated Trp at their core, have been the subject of considerable interest.[22] Our findings therefore expand the chemical diversity of cyanobactins and confirm the existence of a rare Trp prenyltransferase. The prenyltransferases of the cyanobactin family are now known to catalyze the O-, C-, and N-prenylation of amino acids in cyclic peptides.