Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis

Abstract

Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the ars operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III polyketide synthases (PKSs), were unknown. Here, we show that two type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the ars operon, are responsible for the biosynthesis of C(22)-C(26) fatty acids from malonyl-CoA. In vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the C(22)-C(26) fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl-CoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were thus found to be synthesized solely from malonyl-CoA by the four members of the ars operon. This is the first demonstration that a type I FAS interacts directly with a type III PKS through substrate transfer.

Fig. 1.

Phenolic lipid synthesis in A. vinelandii. (A) Structures of alkylresorcinols and alkylpyrones that accumulate in cysts. (B) Organization of the ars operon. (C) A proposed pathway for the biosynthesis of alkylresorcinols and alkylpyrones by the Ars proteins.

Fig. 2.

Fatty acid biosynthesis and domain organization of FASs. (A) Fatty acid biosynthesis by FAS. (B) Domain organization of ArsA and ArsD. Numbers below the sequences indicate the amino acid residues, assigning the N-terminal Met as 1. (C) Domain organization of mammalian type I FASs. (D) Domain organization of α and β subunits of fungal and yeast type I FASs.

Fig. 3.

Chromatography analysis of the products of Ars reactions. (A and B) HPLC chromatograms of lipid extracts prepared from E. coli harboring pETDuet–ArsAD and pACYC–ArsB (A) or pETDuet–ArsAD and pCDF–ArsC (B). (C and D) Negative extracted ion LC-APCIMS chromatograms of in vitro reactions containing ArsA, ArsB, and ArsD (C) and ArsA, ArsC, and ArsD (D).

Fig. 4.

Radio-TLC analysis of in vitro reaction products. (A) Analysis of products produced from malonyl-CoA by ArsA and ArsD. The production of radiolabeled products was observed in the presence of [2-¹⁴C]malonyl-CoA, NADPH, ArsA, and ArsD (lane 1). No radioactivity was seen in the absence of ArsA (lane 2), ArsD (lane 3), or NADPH (lane 4). (B) Analysis of a hydrolyzed reaction mixture containing ArsA and ArsD. After incubation of malonyl-CoA in the presence of ArsA and ArsD, the reaction mixture was hydrolyzed by alkali, extracted with ethyl acetate, and subjected to TLC. Lane 1 is a negative control containing a complete reaction mixture that was not subjected to hydrolysis. [¹⁴C] attached to ArsA was fractionated into the water layer and did not appear in the TLC analysis. Lane 2 contains a complete reaction mixture that was hydrolyzed and extracted with ethyl acetate. Lanes 3 and 4 contain a macromolecular fraction of the reaction mixture that was hydrolyzed (lane 4) or not hydrolyzed (lane 3). Lanes 5 and 6 contain a low-molecular-weight fraction that was hydrolyzed (lane 6) or not hydrolyzed (lane 5). (C) Analysis of the products of ArsA, ArsB, and ArsD from two different starter substrates. The starter substrates used were [2-¹⁴C]acetyl-CoA (lane 1) and [2-¹⁴C]malonyl-CoA (lane 2). Lane 3 contains an authentic sample 1a prepared from behenyl-CoA (C22-CoA) and malonyl-CoA by ArsB. (D) Analysis of the products of ArsA, ArsC, and ArsD from two different starter substrates. The starter substrates used were [2-¹⁴C]acetyl-CoA (lane 1) and [2-¹⁴C]malonyl-CoA (lane 2). Lane 3 contains authentic samples 1b and 1c prepared from behenyl-CoA (C22-CoA) and malonyl-CoA by ArsC.

Fig. 5.

Radiolabeling analysis of the direct transfer of acyl products from ArsA to ArsB (A and B) or ArsC (C and D) by SDS/PAGE (A and C) and radio-TLC (B and D). The substrates used were [¹⁴C]acyl-ArsA (lane 1), [¹⁴C]acyl-ArsA + malonyl-CoA (lane 2), [¹⁴C]acyl-ArsA + ArsB or ArsC (lane 3), [¹⁴C]acyl-ArsA + ArsB or ArsC + malonyl-CoA (lane 4), [¹⁴C]acyl-ArsA + ArsB or ArsC mutants (lane 5), and [¹⁴C]acyl-ArsA + ArsB or ArsC mutants + malonyl-CoA (lane 6). Lane 7 contains 1a (B) or 1b and 1c (D). Production of radiolabeled alkylresorcinol (B) and alkylpyrone (D) was observed (lane 4). The chemical structures of these compounds are shown in Fig. 1A.