The phosphopantetheinyl transferases: catalysis of a post-translational modification crucial for life

Abstract

Covering: up to 2013. Although holo-acyl carrier protein synthase, AcpS, a phosphopantetheinyl transferase (PPTase), was characterized in the 1960s, it was not until the publication of the landmark paper by Lambalot et al. in 1996 that PPTases garnered wide-spread attention being classified as a distinct enzyme superfamily. In the past two decades an increasing number of papers have been published on PPTases ranging from identification, characterization, structure determination, mutagenesis, inhibition, and engineering in synthetic biology. In this review, we comprehensively discuss all current knowledge on this class of enzymes that post-translationally install a 4'-phosphopantetheine arm on various carrier proteins.

Fig. 1

General reaction scheme of post-translational phophopantetheinylation by a PPTase. The PPTase transfers the PPant moiety from CoA to a conserved serine residue on the apo-CP to produce holo-CP, here showcased by a typical NRPS module containing C, condensation; A, adenylation; and CP, carrier protein, domains. 3′, 5′-PAP is 3′,5′-phosphoadenosine phosphate.

Fig. 2

PPTases. Overview of the three families of PPTases, typified by AcpS, Sfp and the integrated PPTase domain of Saccharomyces cerevisiae FAS2, and a sequence alignment of archetypical PPTases using TCoffee and Espript^, (Bs, Bacillus subtilis, Ec, Escherichia coli. Hs, Homo sapiens and Sc, Saccharomyces cerevisiae.)

Fig. 3

Biosynthesis of enterobactin and the corresponding gene cluster including the PPTase entD, labelled in red (GenBank acc. no. NP_415115.2) (data extracted from the E. coli genome with the GenBank acc. no. NC_000913.2). ICL represents isochorismate lyase,; PCP, peptidyl carrier protein; C, condensation domain; A, adenylation domain; and TE, thioesterase domain.

Fig. 4

Comparison of E. coli O157 (top) and E. coli K12 (bottom) in the region of the PPTase acpT (in red).

Fig. 5

Colibactin biosynthetic gene cluster with the PPTase gene clbA (GenBank acc. no. AM229678.1). NRPS genes: yellow, PKS genes: blue, PKS-NRPS hybrid genes: green, PPTase gene: red.

Fig. 6

Secondary metabolites of Streptomyces coelicolor that depend on PPTase activity. Antismash identifies additionally butyrolactone (type I PKS), an NRPS natural product (nrp-cys) and another type I PKS product.^,

Fig. 7

Comparison of the nosiheptide and nocathiacin gene clusters. The adenylation domain is shown in green, the putative carrier protein in blue and the ribosomal pre-peptide in yellow. Sequence alignment of E. coli AcpP and nosJ (ACR48339.1), sioP (ADR01086.1), nocK (ACN52299.1) and tsrP (ACN80653.1) show the conserved (D/T)SL motif, characteristic of a carrier protein. Sequence alignment was made using TCoffee and Espript.^,

Fig. 8

Prodigiosin biosynthesis requires both PPTases PswP and PigL. Prodigiosin biosynthetic gene cluster (GenBank acc. no. AJ833002.1) including the PPTase gene pigL, labelled in red.

Fig. 9

Carboxylic acid reductase (CAR). CAR is a promiscuous enzyme that reduces several (R′=) acyl and aromatic carboxylic acids to an aldehyde. Carboxylic acids are first activated by an adenylation domain (A), loaded onto the carrier protein (CP) and in a NADPH-dependent fashion reduced from the carrier protein by the reductase domain (R). The aldehyde product can further be processed to yield alcohols or alkanes.

Fig. 10

PUFA-PKS biosynthetic gene cluster (GenBank acc. no. EU719604.1) including the PPTase gene pfaE, labelled in red.

Fig. 11

Sequence alignments of archaeabacterial PPTases. Only the active-sites are shown.

Fig. 12

Biosynthetic gene clusters surrounding the three PPTase genes of Nostoc punctiforme PCC 73102 (ATCC 29133) (GenBank acc. no. NC_010628). A: NgcS (GenBank acc. no. YP_001863782) with glycolipid PKS locus, B: PPTase (GenBank acc. no. YP_001865721) with PKS-NRPS locus, C: PPTase (GenBank acc. no. YP_001865651) with PKS locus. Domains identified with Antismash in bold. NRPS genes: yellow, PKS genes: blue, NRPS-PKS hybrid genes: green, PPTase gene: red. A: adenylation domain, ACP: acyl carrier protein, AT: acyl transferase, C: condensation domain, DH: dehydratase, DS: desaturase, E: epimerization domain, ER: enoylreductase, HP: hypothetical protein, KR: ketoreductase, KS: β-ketoacyl synthase, mCAT: malonyl CoA-ACP transacylase, MT: methyl transferase, Ox: NADH:flavin oxidoreductase, P5CR: pyrroline-5-carboxylate reductase, PCP: peptide carrier protein, PKS: polyketide synthase, SDR: short-chain dehydrogenase/reductase, TD: thioester reductase, TE: thioesterase, TP: thiamine pyrophosphate binding domain containing protein.

Fig. 13

Alignment of HetI-like PPTases from Anabaena sp. PCC7120 (A7_HetI), N. punctiforme PCC 73102 (Np_NgcS) and N. spumigena NSOR10 (Ns_NsPPT) against Sfp (Bs_Sfp) (GenBank acc. no. AAA22003, ACC78839, AAW67221, CAA44858). Sequence alignment was made using TCoffee and Espript.^,

Fig. 14

Targets of PPTase activity in fungi, showcased by activities of the PPTase NpgA from A. nidulans.

Fig. 15

Ebony from Drosophila resembles an NRPS and catalyzes the formation of β-alanine-amines, like β-alanine-dopamine. Also other biological amines are used to produce β-alanine-amines. AS represents an amine-selecting domain; A, adenylation domain; and CP a carrier protein.

Fig. 16

Activities of human PPTase AASDHPPT. A) 10-THF involved in 10-fTHF metabolism, B) AASDH involved in catabolism of lysine, C) mitochondrial FAS and D) cytosolic FAS. DH represents a dehydrogenase; A, an adenyltransferase; R, a reductase; ACP, acyl carrier protein; MAT, malonyl-CoA acyltransferase; KS3, ketoacyl synthase III; KR, ketoreductase; DH, dehydratase; ER, enoyl reductase; KS, ketoacyl synthase; and TE, thioesterase.

Fig. 17

Neighbour Joining Method phylogenetic tree of annotated ~60 PPTases (see Table 1), constructed using MEGA.

Fig. 18

Sequence alignment of several carrier proteins from Rhizobia. Ec_acpp is E. coli ACP, NodF a CP involved in lipochitin nodulation factor biosynthesis, rkpF a CP involved in capsular polysaccharide biosynthesis, R1_acpp the AcpP of R. leguminosarum, SM_b20651 and R02304 two other CPs present in R. leguminosarum.

Fig. 19

Left: AcpS-ACP co-crystal structure, showing the B. subtilis AcpS trimer and three B. subtilis ACPs (in orange) binding to the PPTase. (PDB:1F80). Right: zoom in on the interaction between helix I of AcpS and helix III of ACP. Crucial amino acids are labeled.

Fig. 20

Sequence alignment of carrier proteins and peptide mimics. TycA_PCP is the PCP1 from tyrocidine synthase TycA, SrfB_PCP1 is the PCP1 from surfactin synthase SrfB, GrsA_PCP is the PCP from Gramicidin synthase GrsA, EntF and EntB are the PCPs from enterobactin synthase, EcACPP is E. coli AcpP, hACPP is the excised human AcpP; YbbR, peptideS, peptideA and 8mer are the short peptides found to be substrates of PPTases.^{, ,}

Fig. 21

AcpH, ACP hydrolase or ACP phosphodiesterase. A) Reaction catalyzed by AcpH. B) Model of E. coli and P. aeruginosa AcpH (modeled after SpoT). SpoT (PDB: 1VJ7) in grey, EcAcpH in orange and PaAcpH in orange. The natural substrate of SpoT, ppGpp, is shown in sticks and the Mn²⁺ ion as a purple sphere.

Fig. 22

Comparison of B. subtilis AcpS and Sfp. (A) Top view of both enzymes. CoA is colored red, and divalent cations are shown as black spheres. Each trimer of AcpS is colored differently to show arrangement of monomers, and each “pseudodimer” of Sfp half is colored differently for comparison to AcpS. (B) Assignment of secondary structure to both AcpS and Sfp. The α-helices are shown in in blue, and β-sheets shown in orange.)

Fig. 23

Comparison of the active site of B. subtilis AcpS and Sfp. CoA is shown in stick form, divalent cations are shown as black spheres, and important residues are shown in ball-and-stick form. (A). Residues on different monomers are colored green and yellow.(B) The PPant moiety of CoA was removed for clarity.

Fig. 24

Overlay of a monomer of AcpS onto the structure of Sfp, exhibiting the similarity of the pseudodimer to the interface between two AcpS monomers. The “arm” in the C-terminal portion of Sfp has been omitted for clarity. (A) Overlay onto the C-terminal portion of Sfp. Strong secondary structure conservation is observed. (B) While overall shape is conserved, less similarity is observed between AcpS and the N-terminal portion of Sfp.

Fig. 25

Cartoon representation of AASDHPPT. (A) The C-terminal “tail” present in ASDPPT but not in Sfp is depicted in red, with CoA in grey and Mg²⁺ in Black. (B) Human FASI ACP-AASDHPPT co-crystal structure. A Ser to Ala mutation was performed on the ACP to prevent 4′-phosphopantetheinylation. The Ala residue is positioned closed to the phosphate that is attacked by the native Ser. Density for CoA was only observed up to two carbons past the diphosphate.

Fig. 26

CoA Analogs: CoA can be broken down into several parts, including phosphopantetheine, pantetheine, pantothenate, and cysteamine. Sfp and other Sfp-type PPTases can utilize CoA analogs that bear modified pantetheine arms

Fig. 27

Scheme demonstrating the diversity of biotechnological applications of the carrier protein-PPTase interaction.

Fig. 28

Inhibitors of AcpS-type and Sfp-type PPTases. ^{, , ,}