The phosphopantetheinyl transferase superfamily: phylogenetic analysis and functional implications in cyanobacteria

Abstract

Phosphopantetheinyl transferases (PPTs) are a superfamily of essential enzymes required for the synthesis of a wide range of compounds including fatty acid, polyketide, and nonribosomal peptide metabolites. These enzymes activate carrier proteins in specific biosynthetic pathways by the transfer of a phosphopantetheinyl moiety to an invariant serine residue. PPTs display low levels of sequence similarity but can be classified into two major families based on several short motifs. The prototype of the first family is the broad-substrate-range PPT Sfp, which is required for biosynthesis of surfactin in Bacillus subtilis. The second family is typified by the Escherichia coli acyl carrier protein synthase (AcpS). Facilitated by the growing number of genome sequences available for analyses, large-scale phylogenetic studies were utilized in this research to reveal novel subfamily groupings, including two subfamilies within the Sfp-like family. In the present study degenerate oligonucleotide primers were designed for amplification of cyanobacterial PPT gene fragments. Subsequent phylogenetic analyses suggested a unique, function-based PPT type, defined by the PPTs involved in heterocyst differentiation. Evidence supporting this hypothesis was obtained by sequencing the region surrounding the partial Nodularia spumigena PPT gene. The ability to genetically classify PPT function is critical for the engineering of novel compounds utilizing combinatorial biosynthesis techniques. Information regarding cyanobacterial PPTs has important ramifications for the ex situ production of cyanobacterial natural products.

FIG. 1.

PPT activation of apo-carrier proteins. A PPT catalyzes the nucleophilic attack (of the hydroxyl side chain of the conserved carrier protein serine residue) on the 5′-β-pyrophosphate linkage of CoA. This causes the transfer of the phosphopantetheinyl moiety of CoA to the side chain of the conserved serine residue, converting the carrier protein from an inactive apo-form to an active holo-form (26).

FIG. 2.

Box shade alignment of “Sfp-like” PPT family representatives. Black shading displays identical residues, and gray shading depicts similar residues. Two subfamilies are shown. The F/KES (top alignment) and W/KEA (lower alignment) sequences are separated with a consensus (cons) line shown below each subfamily. Sequences include the following: Pse, Pseudomonas aeruginosa, AAG04554; Xan, Xanthomonas albicans, AAG28384; Vib, Vibrio cholerae, AAD48884; Pho, Photorhabdus luminescens, AAK16071; Bac, Bacillus subtilis, P39135; Syn, Synechocystis PCC6803, BAA10326; Cae, Caenorhabditis elegans, A89451; Dro, Drosophila melanogaster, AAM11059. PPT motifs are boxed and numbered, including 1A as described. Numbering for the Sfp PPT from Bacillus subtilis is shown in brackets, and an asterisk indicates residues implicated in stability or activity roles.

FIG. 3.

Phylogenetic analysis of a selection of Sfp-like PPTs. A phylogenetic tree representing Sfp-like family is displayed, with accession numbers given in parenthesis. Significant bootstrap data (over 500 of 1,000 repeats) are shown. The E. coli AcpS was chosen as an outgroup for the Sfp-like PPT clade. Two subgroups are observed and distinguished as the W/KEA and F/KES subfamilies, respectively. Letters in superscript refer to PKS biosynthesis (P), NRPS biosynthesis (N), hybrid PKS/NRPS biosynthesis (H), siderophore biosynthesis (S), sequences obtained through the translation of contiguous sequences from unfinished genome projects (✽) (see the supplemental material), Sfp-like PPTs found in genomes without an AcpS (A-), cyanobacterial PPTs associated with heterocyst glycolipid synthesis (HET), and PPTs associated with lysine biosynthesis (L).

FIG. 4.

A phylogenetic tree of cyanobacterial PPTs. Accession numbers are given in parenthesis, and underlined sequences indicate those isolated during the present study. Heterocyst forming cyanobacteria are indicated in bold type. Significant bootstrap data (greater than 500 out of 1,000 repeats) are shown. The PPT from the photosynthetic green sulfur bacterium Chlorobium tepidium was chosen as an outgroup. Distinct phylotypes are observed and depicted as subgroup A (associated with heterocyst glycolipid synthesis) and B, respectively.