Enzymes: Phosphopantetheine‐dependent

Abstract

Phosphopantetheine‐dependent enzymes are a group of multifunctional or individual enzymes including fatty acid synthases, polyketide synthases and nonribosomal polypeptide synthases, all of which utilize the 4′‐phosphopantetheine moiety attached to an acyl carrier protein or a peptide carrier protein. The terminal thiol group of 4′‐phosphopantetheine is covalently attached to any of a number of acyl groups in the form of a thiol ester to facilitate acyl transfer and nucleophilic activation.

Keywords: 4′‐phosphopentetheine; coenzyme A (CoA); thiol ester; C–C bond formation; acyl carrier protein; fatty acid synthase; nonribosomal polypeptide synthase; polypeptide carrier protein; antibiotic biosynthesis

Figure 1.

Formation of holo‐ACP from coenzyme A and apo‐ACP catalysed by 4′‐phosphopantetheine transferase (holosynthase). An invariant serine of the apo‐ACP interacts with the transferase and CoASH in the presence of Mg2+, allowing the serine hydroxyl group to react with the phosphodiester bond of CoASH, displacing AMP and forming the serine‐4′‐phosphopantetheine linkage. The holo‐ACP cysteamine thiol is then able to form ACP‐thiol esters, normally by exchange with an acyl derivative in the presence of a specific transacylase.

Figure 2.

Chemistry of fatty acid synthases, polyketide synthase and nonribosomal polypeptide synthases. In fatty acid synthases and polyketide synthases an electrophilic acyl‐CoA starter or 4′‐phosphopantetheine‐bound acyl thiol ester is transferred to a reactive cysteine residue of a condensing enzyme (KS). This then reacts with a nucleophilic α‐carbon atom of malonyl‐ACP accompanied by decarboxylation (a). The resulting β‐ketoacyl thiol ester is then processed according to the specificity of the enzyme (see Figures and text). In some polyketide synthases, methylmalonyl‐ACP may be used. The thiol ester also has an important influence during the subsequent manipulations of 4′‐phosphopantetheine‐bound intermediates, facilitating protonation at the β position of the β‐ketoacyl, enoyl and β‐hydroxy intermediates during the reductions and dehydration steps. In nonribosomal polypeptide synthases (b) it is the aminoacyl thiol ester (R1) attached to a peptidyl carrier protein PCPn−1 that acts as the electrophile, the nucleophile being the amino acid of the aminoacyl‐PCPn thiol ester (R2). The reaction is mediated by a specific condensation domain (C). The dipeptide, now attached to PCPn, reacts with a third aminoacyl‐PCPn+1 carrying an amino acid (R3). See also Figure .

Figure 3.

Generalized scheme for fatty acid synthesis. Initially, in a priming reaction, the acetyl group of acetyl‐CoA is transferred to the acyl carrier protein (ACP) from which it is then transferred to a cysteine thiol of β‐ketoacyl synthase (KS). The malonyl moiety of malonyl‐CoA is then linked to the vacant ACP thiol by a malonyltransferase (MT) and condensation occurs, with decarboxylation, to give the acetoacetyl‐thiol ester of ACP. All subsequent reactions occur with the intermediates bound through a thiol ester link to the ACP thiol. Reduction by NADPH is catalysed by the ketoreductase (KR) to give a secondary alcohol, followed by dehydration, catalysed by dehydratase (DH). A second reduction by NADPH, catalysed by enoyl reductase (ER) yields the fully saturated butyryl‐ACP thiol ester. The butyryl moiety is then transferred to the KS active site thiol to complete the ‘cycle’. Subsequent cycles occur as above, each commencing with the loading of the ACP with a new malonyl moiety. The cycles continue until a C16 chain has been assembled, at which point the fatty acid is released from the ACP by a thioesterase (TE). The thioesterase is specific for a chain length of 16 carbon atoms.

Figure 4.

Organization of functionalities within the dimeric chicken liver fatty acid synthase. Proteolytic studies have revealed that the chicken FAS is composed of three main ‘domains’ – I, II and III. Domain I contains the KS and MT/AT functionalities; domain II contains the DH, ER, KR, and ACP functions and domain III is the thioesterase (TE). The enzyme functions as a dimer with the active‐site cysteine of one KS functionality being close to the 4′‐phosphopantetheine cofactor of the ACP from the other subunit in a head‐to‐tail arrangement.

Figure 5.

Structure and active site of the β‐ketoacyl synthase (KS) from Escherichia coli. Ribbon diagram of the β‐ketoacyl synthase II from E. coli (PDB access code 1KAS). The interrelationship between the side‐chains of the active‐site residues Cys163, His303 and His340 are shown. See text for details.

Figure 6.

Active‐site residues of malonyl‐CoA:ACP transacylase from Escherichia coli. The three‐dimensional arrangement of the catalytic dyad (Ser/His) in the E. coli malonyl‐CoA:ACP transacylase (PDB code 1MLA). Gln250 stabilizes the Nδ‐1 of His201 and replaces the aspartate, typically seen in Asp/His/Ser triads. The side‐arm of Arg117 is also shown, and the Nε‐2 protons of Arg117 hydrogen‐bond to the side‐chain hydroxyl of the nucleophilic Ser92. Residues Ser92, His201 and Arg117 constitute three out of eight invariant residues in homologues of malonyl‐CoA:ACP transacylase.

Figure 7.

Solution structures of the fatty acid synthase ACP from E. coli. Two distinct solution structures ((a) and (b)) determined for the Esherichia coli fatty acid synthase ACP. ACP1 shows a solution in which the secondary structure is seen to be largely intact, whereas ACP2 shows significant disruption of the helices. In both cases, the solutions produce a subset of violations of the nuclear magnetic resonance‐derived distance data used to derive the final structures. It is thought that significant conformational averaging means there are necessarily several possible solution structures.

Figure 8.

Polyketide derived secondary metabolites of commercial importance.

Figure 9.

Scheme for classical and reduced processive polyketide synthesis.

Figure 10.

Diagramatic representation of the modules of 6‐deoxyerythronolide B synthase from Saccharopolyspora erythraea. The enzyme consists of three giant proteins, DEBS‐1, DEBS‐2 and DEBS‐3, each of which is made up of two modules. Each module contains all the necessary functionalities for the condensation and appropriate processing of each methylmalonyl extender unit. Thus module 4 contains a full set of functionalities – KS, AT, DH, ER, KR and ACP – arranged as in fatty acid synthases, with KR out of sequence. Module 1 also contains an additional AT and ACP for loading the starter propionyl unit. Module 6 contains a thioesterase (TE) responsible for releasing the product from the terminal ACP.

Figure 11.

Formation of the ‘unnatural’ polyketides SEK4 and SEK4b, in vivo, using genetically engineered PKS from Streptomyces coelicolor. All the components of the act locus from S. coelicolor (see insert) function to synthesize actinorhodin. Use of a minimal PKS (actI) containing only β‐ketoacyl synthase (KS), chain length factor (CLF) and ACP results in the octaketides SEK4 and SEK4b. Addition of the remaining genes leads to a variety of other unnatural polyketides. For instance, the addition of the ketoreductase (KR) leads to the formation of a different octaketide, mutactin. The presence of the aromatase (ARO) gives rise to SEK34 and the presence of cyclase (CYC) leads to 3,8‐dihydroxy‐1‐methylanthraquinone‐2‐carboxylic acid (DMAC) and aloesaponarin.

Figure 12.

The mechanistic role of 4′‐phosphopantetheine in the peptide carrier protein (PCP) from a nonribosomal polypeptide synthase (NRPS) and the arrangement of the functionalities of surfactin synthase. (a) Stages in the formation of a peptide bond by a nonribosomal polypeptide synthase (NRPS). The carboxyl group of the amino acid, R, is activated as its acyladenylyl derivative and linked to the 4′‐phosphopantetheine arm of a specific PCPn by a highly specific acyltransferase (A). The free amino group of this aminoacyl‐PCP extender then attacks the thiol ester bond of the previous amino acid (or peptide), already bound as its aminoacyl‐PCPn−1 by a similar activation process. The reaction, catalysed by a specific condensing enzyme (C), leads to the formation of the new peptide bond and the displacement of the PCPn−1. The peptide, still linked to PCPn, can be further elongated by reaction with another amino group from an aminoacyl‐PCPn+1 (not shown). In this way, the peptide is built in a stepwise process, from N‐terminus to C‐terminus, passing along the PCP functionalities until the final stage when the peptide‐PCP thiol ester bond is hydrolysed to yield the terminal carboxyl group, releasing the product from the enzyme (not shown). Alternatively, the terminal thiol ester may be displaced by the amino group of the N‐terminal amino acid to form a cyclic peptide. (b) Arrangement of the functionalities of the NRPS surfactin synthase. The synthase is arranged in modules, each with condensing enzyme (C), acyltransferase (A) and peptide carrier protein (PCP) functionalities and the additional possibility of an epimerase (EPI) functionality. Modules, one for the addition of each amino acid, are arranged in the sequence of amino acids in the final peptide chain. Thus, the first module links glutamate to the C12 starter unit, the second links leucine, and so on. The energy for the final cyclization to form the ester comes from the reaction of the hydroxyl group with the terminal aminoacyl‐PCP thiol ester.

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References

Abell C and Staunton J (1984) Biosyntheis of 6‐methylsalicylic acid – the combined use of mono‐deuteriated and tri‐deuteriated acetate precursors to investigate the degree of stereocontrol in the aromatisation sequence. Journal of the Chemical Society, Chemical Communications 15: 1005–1007.

Andrec M, Hill RB and Prestegard JH (1995) Amide exchange‐rates in E. coli acyl carrier protein – correlation with protein structure dynamics. Protein Science 4: 983–993.

Aparicio JF, Molnar I, Schwecke T et al. (1996) Organisation of the biosynthetic gene cluster fpr rapamycin in Streptomyces hygroscopius: analysis of the enzymatic domains in the modular polyketide synthase. Gene 169: 9–16.

Arnstadt K‐I, Schindlbeck G and Lynen F (1975) Zum Mechanismus der Kondensationreaktion de Fettsaürebiosynthese. European Journal of Biochemistry 55: 561–571.

Baldock C, Rafferty JB, Stuitfe AR, Slabas AR and Rice DW (1998) The X‐ray structure of Escherichia coli enoyl reductase with bound NAD+ at 2.1 Å resolution. Journal of Molecular Biology 284: 1529–1546.

Bayan N and Therisod H (1989) Evidence for interactions of acyl carrier protein with glycerol‐3‐phosphate acyltransferase, an inner membrane protein of E. coli. FEBS Letters 255: 330–334.

Beck J, Ripka S, Siegner A, Schiltz E and Schweizer E (1990) The multifunctional 6‐methylsalicyclic acid synthase gene of Penicillium patulum: its gene structure relative to that of other polyketide synthases. European Journal of Biochemistry 192: 487–498.

Birch AJ and Donovan FW (1953) Studies in relation to biosynthesis. I. Some possible routes to derivatives of orcinol and phloroglucinol. Australian Journal of Chemistry 6: 360–368.

Cane DE, Lambalot RH, Prabhakaran PC and Ott WR (1993) Macrolide biosynthesis. 7. Incorporation of polyketide chain elongation intermediates into methymycin. Journal of the American Chemical Society 115: 522–526.

Coderre PE and Earhart CF (1989) The EntD gene of the E. coli K12 enterobactin gene cluster. Journal of General Microbiology 135: 3043–3055.

Cortes J, Haydock SE, Roberts GA, Bevitt DJ and Leadlay PF (1990) An unusually large multifunctional polypeptide in the erythromycin‐producing polyketide synthase of Saccharopolyspora erythrea. Nature 348: 176–178.

Crump MP, Crosby J, Dempsey CE et al. (1997) Solution structure of the actinorhodin polyketide synthase acyl carrier protein from Streptomyces coelicolor A3(2). Biochemistry 36: 6000–6008.

Donadio S, McAlpine JB, Sheldon PJ, Jackson M and Katz L (1993) An erythromycin analog produced by reprogramming of polyketide synthesis. Proceedings of the National Academy of Sciences of the USA 90: 7119–7123.

Fischl AS and Kennedy EP (1990) Isolation and properties of acyl carrier protein phosphodiesterase of E. coli. Journal of Bacteriology 172: 5445–5449.

Fu H, Ebert‐Khosla S, Hopwood DA and Khosla C (1994a) Engineered biosynthesis of novel polyketides: dissection of the catalytic specificity of the act ketoreductase. Journal of the American Chemical Society 116: 4166–4170.

Fu H, Hopwood DA and Khosla C (1994b) Engineered biosynthesis of novel polyketides: evidence for temporal, but not regiospecific control of cyclisation of an aromatic polyketide precursor. Chemistry and Biology 1: 205–210.

Hitchman TS, Crosby J, Byrom KJ, Cox RJ and Simpson TJ (1998) Catalytic self‐acetylation of type II polyketide synthase acyl carrier proteins. Chemistry and Biology 5: 35–47.

Hopwood DA (1997) Genetic contributions to understanding polyketide synthases. Chemical Review 97: 2465–2497.

Hopwood DA and Sherman DH (1990) Molecular genetics of polyketides and its comparison to fatty acid biosynthesis. Annual Review of Genetics 24: 37–66.

Huang W, Jia J, Edwards P, Dehesh K, Scheider G and Lindquist Y (1998) Crystal structure of β‐ketoacyl‐acyl carrier protein synthase II from E. coli reveals the molecular architecture of condensing enzymes. EMBO Journal 17: 1183–1191.

Jacobsen JR, Hutchinson CR, Cane DE, Khosla C (1997) Precursor‐directed biosynthesis of erythromycin analogs by an engineered polyketide synthase. Science 277: 367–369.

Jordan PM and Spencer JB (1991) Mechanistic and stereochemical investigation of fatty acid and polyketide biosynthesis using chiral malonates. Tetrahedron 47: 6015–6028.

Khosla C, Ebert‐Khosla S and Hopwood DA (1992) Targeted gene replacements in a Streptomyces polyketide synthase gene cluster – role for the acyl carrier protein. Molecular Microbiology 6: 3237–3249.

Khosla C, McDaniel R, Ebert‐Khosla S et al. (1993) Genetic construction and functional analysis of hybrid polyketide synthases containing heterologous acyl carrier proteins. Journal of Bacteriology 175: 2197–2204.

Kim Y and Prestegard JH (1990) Refinement of the NMR structures for acyl carrier protein with scalar coupling data. Proteins 8: 377–385.

Lambalot RH and Walsh CT (1995) Cloning, overproduction, and characterisation of the Escherichia coli Holo‐acyl carrier protein synthase. Journal of Biological Chemistry 270: 24658–24661.

Lambalot RH, Gehring AM, Flugel RS et al. (1996) A new enzyme superfamily – the phosphopantetheinyl transferases. Chemistry and Biology 3: 923–936.

Lanz T, Tropf S, Marner F‐J and Schröder J (1991) The role of cysteines in polyketide synthases. Journal of Biological Chemistry 266: 9971–9976.

Lynen F (1967) Biosynthesis pathways from acetate to natural products. Pure and Applied Chemistry 14: 137–159.

McCarthy AD and Hardie DG (1984) Fatty acid synthase – an example of protein evolution by gene fusion. Trends in Biochemical Sciences 9: 60–63.

McDaniel R, Ebert‐Khosla S, Hopwood DA and Khosla C (1993a) Engineered biosynthesis of novel polyketides. Science 262: 1546–1550.

McDaniel R, Ebert‐Khosla S, Hopwood DA and Khosla C (1993b) Engineered biosynthesis of novel polyketides: manipulation and analysis of an aromatic polyketide synthase with unproven catalytic specificities. Journal of the American Chemical Society 115: 11671–11675.

McDaniel R, Ebert‐Khosla S, Hopwood DA and Khosla C (1994a) Engineered biosynthesis of novel polyketides: act VII and act IV genes encode aromatase and cyclase enzymes respectively. Journal of the American Chemical Society 116: 10855–10859.

McDaniel R, Ebert‐Khosla S, Fu H, Hopwood DA and Khosla C (1994b) Engineered biosynthesis of novel polyketides: influence of a downstream enzyme on the catalytic specificity of a minimal PKS. Proceedings of the National Academy of Sciences of the USA 91: 11542–11546.

McDaniel R, Hutchinson CR and Khosla C (1995a) Engineered biosynthesis of novel polyketides: analysis of TcmN function in tetracenomycin biosynthesis. Journal of the American Chemical Society 117: 6805–6810.

McDaniel R, Ebert‐Khosla S, Hopwood DA and Khosla C (1995b) Rational design of aromatic polyketide natural products by recombinant assembly of enzymatic subunits. Nature 375: 549–554.

Nakano MM, Corbell N, Besson J and Zuber P (1992) Isolation and characterisation of sfp: a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in Bacillus subtilis. Molecular and General Genetics 232: 313–321.

O'Hagan D (1991) The Polyketide Metabolites. Chichester, UK: Ellis Horwood.

O'Hagan D (1995) Biosynthesis of fatty‐acid and polyketide metabolites. Natural Product Reports 12: 1–32.

Perham RN (2000) Swinging arms and swinging domains in multifunctional enzymes: catalytic machines for multistep reactions. Annual Review of Biochemistry 69: 961–1004.

Polacco ML and Cronan JE (1981) A mutant of E. coli conditionally defective in the synthesis of holo‐acyl carrier protein. Journal of Biological Chemistry 11: 5750–5754.

Prescott DJ and Vagelos PR (1972) Acyl carrier protein. Advances in Enzymology and Related Areas of Molecular Biology 36: 269–311.

Prescott DJ, Elovson J and Vaglos PR (1969) Acyl carrier protein. Journal of Biological Chemistry 244: 4517–4521.

Sedgwick B, Cornforth JW, French SJ, Gray RT, Kelstrup E and Willadsen P (1977) The biosynthesis of long‐chain fatty acids. Incorporation of radioactivity from stereospecifically tritiated malonyl thiol esters and the stereochemistry of the acetyl‐CoA carboxylase reaction. European Journal of Biochemistry 75: 481–495.

Serre L, Verbree EC, Dauter Z, Stuitje AR and Derewenda ZS (1995) The E. coli malonyl‐CoA:acyl carrier protein transacylase at 1.5 Å resolution. Journal of Biological Chemistry 270: 12961–12964.

Shen B and Hutchinson CR (1993) Enzymatic synthesis of a bacterial polyketide from acetyl and malonyl coenzyme A. Science 262: 1535–1540.

Wakil SJ (1989) Fatty acid synthase, a proficient multifunctional enzyme. Biochemistry 28: 4523–4530.

Wakil SJ, Stoops JK and Joshi VC (1983) Fatty acid synthesis and its regulation. Annual Review of Biochemistry 52: 537–579.

Witkowski A, Rangan VS, Randhawa ZL, Amy CM and Smith S (1991) Structural organisation of the multifunctional animal fatty acid synthase. European Journal of Biochemistry 198: 571–579.

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Crump, Matthew P, and Shoolingin‐Jordan, Peter M(Mar 2002) Enzymes: Phosphopantetheine‐dependent. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0003742]