Genetic variation of adenylation domains of the anabaenopeptin synthesis operon and evolution of substrate promiscuity

Abstract

Anabaenopeptins (AP) are bioactive cyclic hexapeptides synthesized nonribosomally in cyanobacteria. APs are characterized by several conserved motifs, including the ureido bond, N-methylation in position 5, and d-Lys in position 2. All other positions of the AP molecule are variable, resulting in numerous structural variants. We have identified a nonribosomal peptide synthetase (NRPS) operon from Planktothrix agardhii strain CYA126/8 consisting of five genes (apnA to apnE) encoding six NRPS modules and have confirmed its role in AP synthesis by the generation of a mutant via insertional inactivation of apnC. In order to correlate the genetic diversity among adenylation domains (A domains) with AP structure variation, we sequenced the A domains of all six NRPS modules from seven Planktothrix strains differing in the production of AP congeners. It is remarkable that single strains coproduce APs bearing either of the chemically divergent amino acids Arg and Tyr in exocyclic position 1. Since the A domain of the initiation module (the ApnA A₁ domain) has been proposed to activate the amino acid incorporated into exocyclic position 1, we decided to analyze this domain both biochemically and phylogenetically. Only ApnA A₁ enzymes from strains producing AP molecules containing Arg or Tyr in position 1 were found to activate these two chemically divergent amino acids in vitro. Phylogenetic analysis of apn A domain sequences revealed that strains with a promiscuous ApnA A₁ domain are derived from an ancestor that activates only Arg. Surprisingly, positive selection appears to affect only three codons within the apnA A₁ gene, suggesting that this remarkable promiscuity has evolved from point mutations only.

Fig. 1.

(A) Scheme of the structural organization of the anabaenopeptin (apn) biosynthetic gene cluster from Planktothrix agardhii strain CYA126/8. The ApnA A₁ domain (boxed) was probed using the ATP-pyrophosphate exchange assay. (B) Chemical structures of anabaenopeptin variants (AP 908 and AP 915) produced by the same strain (34).

Fig. 2.

(A) Inactivation of the apnC gene by insertional inactivation via homologous recombination. The transformation construct contains a selection marker (Cm^r; 800 bp) (shaded) that is flanked by homologous sequences on both the 5′ and 3′ ends. KO, knockout. (B) Testing of the full segregation of the apnC mutation by PCR using primers specific to the flanking region of the construct inserted into apnC (sequence position 15074 in EMBL/GenBank/DDBJ accession no. EF672686). (C) HPLC analysis of the aqueous-methanol extracts of WT apnC (top) and KO apnC, deficient in AP synthesis (bottom). Peaks 1 and 2 represent aeruginosides 126A (m/z 691; 9.07 min) and 126B (m/z 715; 10.56 min); peak 3, microviridin K (m/z 1,771; 16.09 min); peaks 4 and 5, AP 908 (m/z 909; 18.29 min) and AP 915 (m/z 916; 22.67 min); and peaks 6 and 7, MC-RR (m/z 1,024; 26.61 min) and MC-LR (m/z 981; 33.83 min), respectively.

Fig. 3.

Phylogenetic tree of the six A domains (core motifs A1 to A10; 1,441 bp) of the apn gene cluster sequenced from the seven Planktothrix strains investigated. The significant bootstrap percentages were obtained from 1,000 pseudoreplicates using neighbor joining/parsimony/maximum likelihood. For each clade, the genetic dissimilarity was calculated. In addition, the position and identity of each amino acid (AA) incorporated into one of the six positions of the AP molecule are given. The NMT region (1,175 bp) of the apnC A₂ domain was not included in the phylogenetic analysis. The mcyB A₁ sequence of Microcystis strain PCC7806 served as an outgroup. Underlining indicates A domain genetic variants that were probed using the ATP-pyrophosphate exchange assay.

Fig. 4.

Comparison of amino acid activation between ApnA A₁ domains expressed from seven different Planktothrix strains by using the ATP-pyrophosphate exchange assay. Blank 1 contains protein; blank 2 does not.