PROSITE documentation PDOC00012

Carrier protein (CP) domain profile and phosphopantetheine attachment site signature

Carrier protein (CP) domains are crucial components involved in the transfer of thiol ester-bound intermediates during the biosynthesis of primary and secondary metabolites such as fatty acids, polyketides, and nonribosomal peptides. CP domains, also referred to as thiolation domains, are responsible for transporting the substrate and chain intermediates to the catalytic centers of the polyketide synthase (PKS), nonribosomal peptide synthetase (NRPS), and fatty acid synthase (FAS) assembly lines. The biosynthetic chain intermediates are tethered as thioesters on the terminal thiol of a phosphopantetheine (or pantetheine 4' phosphate) (Ppant) prosthetic group that is covalently attached to an invariant serine residue of the CP domain. CP domains, typically consisting of 70-100 residues, are either freestanding or embedded in these multifunctional proteins and exist as three variants: an acyl carrier protein (ACP) found in PKSs and FASs, a peptidyl carrier protein (PCP) found in NRPS systems, and an aryl carrier protein (ArCP) commonly found in siderophore NRPS synthetases [1,2,3]. CP domains have been found in various enzyme systems which are listed below:

• Fatty acid synthase (FAS), which catalyzes the formation of long-chain fatty acids from acetyl-CoA, malonyl-CoA and NADPH. Bacterial and plant chloroplast FAS are composed of eight separate subunits which correspond to the different enzymatic activities; CP is one of these polypeptides. Fungal FAS consists of two multifunctional proteins, FAS1 and FAS2; the CP domain is located in the N-terminal section of FAS2. Vertebrate FAS consists of a single multifunctional enzyme; the CP domain is located between the β- ketoacyl reductase domain and the C-terminal thioesterase domain [4].
• Polyketide antibiotics synthase enzyme systems. Polyketides are secondary metabolites produced from simple fatty acids, by microorganisms and plants. CP is one of the polypeptidic components involved in the biosynthesis of Streptomyces polyketide antibiotics actinorhodin, curamycin, granatacin, monensin, oxytetracycline and tetracenomycin C.
• Bacillus subtilis putative polyketide synthases pksK, pksL and pksM which respectively contain three, five and one CP domains.
• The multifunctional 6-methysalicylic acid synthase (MSAS) from Penicillium patulum. This is a multifunctional enzyme involved in the biosynthesis of a polyketide antibiotic and which contains a CP domain in the C-terminal extremity.
• Multifunctional mycocerosic acid synthase (gene mas) from Mycobacterium bovis.
• Gramicidin S synthase I (gene grsA) from Bacillus brevis. This enzyme catalyzes the first step in the biosynthesis of the cyclic antibiotic gramicidin S.
• Tyrocidine synthase I (gene tycA) from Bacillus brevis. The reaction carried out by tycA is identical to that catalyzed by grsA.
• Gramicidin S synthase II (gene grsB) from Bacillus brevis. This enzyme is a multifunctional protein that activates and polymerizes proline, valine, ornithine and leucine. GrsB contains four CP domains.
• Erythronolide synthase proteins 1, 2 and 3 from Saccharopolyspora erythraea which is involved in the biosynthesis of the polyketide antibiotic erythromicin. Each of these proteins contains two CP domains.
• Conidial green pigment synthase from Aspergillus nidulans.
• ACV synthase from various fungi. This enzyme catalyzes the first step in the biosynthesis of penicillin and cephalosporin. It contains three CP domains.
• Enterobactin synthase component F (gene entF) from Escherichia coli. This enzyme is involved in the ATP-dependent activation of serine during enterobactin (enterochelin) biosynthesis.
• Cyclic peptide antibiotic surfactin synthase subunits 1, 2 and 3 from Bacillus subtilis. Subunits 1 and 2 contain three related domains while subunit 3 only contains a single domain.
• HC-toxin synthase (gene HTS1) from Cochliobolus carbonum. This enzyme synthesizes HC-toxin, a cyclic tetrapeptide. HTS1 contains four CP domains.
• Fungal mitochondrial ACP, which is part of the respiratory chain NADH dehydrogenase (complex I).
• Rhizobium nodulation protein nodF, which probably acts as an CP in the synthesis of the nodulation Nod factor fatty acyl chain.

The CP domain fold is variably referred to as a 3- or 4-helix bundle and typically consists of 3 major α helices, with helix I being antiparallel to helices II and IV, plus a short 3(10) or α-helical segment (helix III) linking helices II and IV (see <PDB:1ACP>) [3].

The sequence around the phosphopantetheine attachment site is conserved in all these proteins and can be used as a signature pattern. A profile was also developed that spans the complete CP domain.