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Synthesis of an affinity alkylating non steroidal mimic of 1alpha,25-dihydroxyvitamin D(3) and its radiolabeled counterpart is presented. We also report the affinity labeling of the VDR-ligand binding domain (VDR-LBD) with this analogue.
4-Oxalocrotonate tautomerase (EC 5.3.2-; 4-OT), a hexamer consisting of 62 residues per subunit, catalyzes the isomerization of unsaturated alpha-keto acids, converting unconjugated ketones to the conjugated isomers via a dienolic intermediate. The recently solved crystal structure of an isozyme of 4-OT suggests that the amino-terminal proline is the catalytic base [Subramanya, H. S., Roper, D. I., Dauter, Z., Dodson, E. J., Davies, G. J., Wilson, K. S., & Wigley, D. B. (1996) Biochemistry 35, 792-802]. In support of this proposed role, we have found that the active-site-directed irreversible inhibitor 3-bromopyruvate (3-BP) blocks the amino terminus of 4-OT to Edman degradation and results in the disappearance of the 15N resonance of Pro-1 (delta = 49.2 ppm at pH 6.40 and 42 degrees C) in the 15N NMR spectrum of uniformly 15N-labeled 4-OT. Furthermore, covalent bonding between a 15N resonance of 4-OT and the methylene carbon of the reduced, 3-(13)C-labeled lactyl adduct derived from [3-(13)C]-bromopyruvate was then directly demonstrated using two heteronuclear NMR methods, an 1H-(13)C HSQC experiment and a novel inverse correlation experiment which we call H(C)N. The chemical shift of the modified 15N resonance (delta = 86.5 ppm) is consistent with that of an alkylated and cationic, amino-terminal proline. Affinity labeling with 2-(14)C-labeled bromopyruvate indicates that the ultimate stoichiometry of modification is I equiv of 3-BP per 4-OT monomer. However, an analysis of the residual enzyme activity after differing extents of fractional modification with 3-BP indicates that modification of three active sites per hexamer abolishes essentially all activity of the hexamer. Thus, 4-OT exhibits half-of-the-sites stoichiometry with 3-BP. Finally, the pH dependence of kinact/KI for affinity labeling by 3-BP yields a pKa value of 6.7 +/- 0.3, in reasonable agreement with the pKa values found for kcat/KM for the non-sticky substrate 2-hydroxy-2,4-pentadienoate and by direct NMR titration of Pro-1 [Stivers, J. T., Abeygunawardana, C., Mildvan, A. S., Hajipour, G., & Whitman, C. P. (1996) Biochemistry 35, 814-823]. These results strongly implicate the amino-terminal proline as the general-base catalyst on 4-OT.
Naloxone benzoylhydrazone (NalBzoH) labels both mu and kappa receptors in standard homogenate binding assays. We now report that [3H]NalBzoH can effectively photoaffinity label opioid receptors. By modifying [3H]NalBzoH binding conditions, we can selectively label either mu or kappa 3 receptors in calf striatal membranes or classical U50,488-sensitive kappa 1 receptors in guinea pig cerebellar membranes. After removal of unbound radioligand, the [3H]NalBzoH-labeled membranes were irradiated with UV light to couple the bound radioligand to its binding site. No specific mu, kappa 1, or kappa 3 binding remained after a 20-hr dissociation at 25 degrees without UV irradiation. In contrast, approximately 45% of mu and 40% of kappa 1 and kappa 3 binding remained after 2 min of UV exposure. In time course studies, increasing the UV exposure from 30 sec to 3 min produced a progressive increase in radioligand incorporation, which did not increase further with UV exposure times up to 5 min. A portion of the binding in irradiated membranes appeared to be covalently coupled to proteins. Following solubilization of irradiated membranes with sodium dodecyl sulfate, approximately 30-40% of the specifically bound radioactivity precipitated with trichloroacetic acid (TCA). These levels of TCA-precipitable binding corresponded to the amount of dissociation-resistant binding described above. No specific binding could be TCA precipitated in samples that were not exposed to UV. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]NalBzoH-labeled membranes revealed a number of labeled peaks with levels of radioactivity that did not correspond to the intensity of the protein bands, suggesting that this technique was not randomly labeling proteins. This approach may be a useful method to affinity label, characterize, and purify mu and kappa opiate receptor subtypes.
Na+/K(+)-ATPase from pig kidney is inactivated by protein-reactive N-hydroxysuccinimidyl derivatives of digoxigenin. Like digoxigenin, its protein-reactive derivatives N-hydroxysuccinimidyl digoxigenin-3-methylcarbonyl-epsilon-aminocaproate (HDMA), 3-amino-3-deoxydigoxigenin hemisuccinimide succinimidyl ester (ADHS), 3-iodoacetylamino-3-deoxydigoxigenin (IAD) and digoxigenin-3-O-succinyl-[2-(N-maleimido)]ethylamide (DSME) inhibited the sodium pump in the presence of Na+, Mg2+ and ATP. At 37 degrees C, half-maximal inhibition of Na+/K(+)-ATPase was seen by HDMA at 0.47 microM, by ADHS at 5.8 microM, by IAD at 8 microM and by DSME at 94 microM. Thus, all compounds bind to the cardiac steroid receptor site of Na+/K(+)-ATPase. Affinity labeling of the alpha subunit by 'front door' or 'back door' phosphorylation was only seen with HDMA or ADHS in the range 0.1 microM. Excess of ouabain protected against affinity labeling. All the other protein-reactive derivatives of digoxigenin labeled the enzyme independent of the formation of a phosphointermediate at much higher concentrations. This labeling was not suppressed by an excess of ouabain. Tryptic hydrolysis of the HDMA-modified Na+/K(+)-ATPase gave peptides of the apparent molecular masses 20, 12.5 and 11.2 kDa. The 11.2-kDa and 12.5-kDa peptides started amino-terminally with Asp68, and the 20-kDa peptide with Asp24. Thus, the HDMA-labeled peptides originate from the cardioactive steroid-binding site formed by the first and second transmembrane helix. N-Hydroxysuccinimidyl esters such as HDMA are normally thought to modify lysine and arginine residues covalently. Since such residues do not exist in the putative cardiac glycoside-binding site, the possibility of a thioester formation of the digoxigenin derivatives HDMA and ADHS with Cys104 in the H1 transmembrane domain was tested. In fact, hydroxylaminolysis led to the release of the covalently bound HDMA, and the formation of a free sulfhydryl group. This could be labeled by [2-14C]ICH2COOH. We therefore propose, consistent with a recent conclusion from a site-directed mutagenesis experiment [Canessa, C. M., Horisberger, J.-D., Louvard, D. & Rossier, B. C. (1992) EMBO J. 11, 1681-1687], that a cysteine residue (probably Cys104) participates in the structure and function of the cardiac glycoside binding.
The trinucleotide AUG was condensed at the 5'-end with N-bromoacetyl-p-aminophenylphosphate. This bromoactylated AUG analog reacted irreversibly with the mRNA binding site of Escherichia coli 70S ribosomes. After reaction of 70S ribosomes with the AUG analog, labeled 30S subunits could be isolated that were programmed for initiation-factor-dependent binding of fMet-tRNAfMet. This shows that this AUG-affinity label reacted in the decoding site for fMet-tRNAfMet. By combination of sodium dodecyl sulfate-, Sarkosyl-, and ureapolyacrylamide gel electrophoresis the AUG-affinity label was found to be irreversibly bound to ribosomal proteins S4, the ram gene product, and S18.
Undegraded dihydropyridine (DHP)-receptor (putatively a voltage-gated Ca2+ channel) has been purified as a 340-kDa protein complex to approximately 80% homogeneity (2.4 nmol of DHP-receptor per mg of protein) from rabbit skeletal muscle by a rapid purification protocol. Transverse-tubule membranes were prepared in high yield by Ribi-press treatment. The DHP-receptor complex was solubilized in 1% digitonin followed by a two-step chromatographic purification procedure. The equilibrium dissociation constant of [3H](+)-PN200-110 binding (Kd; 0.9 nM) was not significantly changed by solubilization or purification. The purified DHP-receptor is composed of two subunits with apparent molecular masses of 148 kDa and 195 kDa migrating in polyacrylamide gels under nonreducing conditions as a single moiety of approximately 300 kDa. The 195-kDa subunit was affinity-labeled with [3H]azidopine in both transverse-tubule membranes and purified DHP-receptor preparations. The subunit can be degraded by high-energy irradiation to a 26-kDa peptide and by proteolysis to a 32-kDa peptide. Thus, it is probably due to proteolytic cleavage and/or photolysis that neither purification nor affinity-labeling studies have previously identified a DHP-receptor subunit of comparable molecular mass (195 kDa).
Slow inward calcium channels in canine cardiac membranes were affinity labeled with the calcium channel analogue, [3H]o-NCS [2,6 dimethyl-3,5-dicarbomethoxy-4-(2- isothiocyanatophenyl )-1, 4-dihydropyridine], in the presence and absence of cold o-NCS or nicardipine. A major specifically labeled peak was identified with Mr 42,000 on NaDodSO4 polyacrylamide gels. In parallel experiments the effects of the calcium channel antagonist, nitrendipine and a variety of other chemical mediators were tested for their ability to stimulate protein phosphorylation in cardiac membranes. These data demonstrate that both nitrendipine and isoproterenol induce the phosphorylation of a 42,000 dalton protein via a kinase endogenous to the cardiac membranes and that the effects of isoproterenol are attenuated by carbachol.
The plasmid pSCR1 containing the gene for mandelate racemase (EC 5.1.2.2) from Pseudomonas putida (ATCC 12633) allows Pseudomonas aeruginosa (ATCC 15692) to grow on (R)-mandelate as its sole carbon source [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540]; the chromosome of the P. aeruginosa host apparently does not contain the gene for mandelate racemase but does contain genes for the remaining enzymes in the mandelate pathway and enables growth on (S)-mandelate as carbon source. However, in the presence of alpha-phenylglycidate, an active-site-directed irreversible inhibitor (affinity label) of mandelate racemase, P. aeruginosa transformed with pSCR1 can utilize (S)-mandelate but not (R)-mandelate as carbon source. This inhibition of growth on (R)-mandelate provides a metabolic selection for mutants that are resistant to alpha-phenylglycidate. When (R)-mandelate is used as carbon source and alpha-phenylglycidate is present, a few colonies of P. aeruginosa transformed with pSCR1 grow slowly and appear on plates after several days. The plasmid isolated from these cells confers resistance to alpha-phenylglycidate on newly transformed cells of P. aeruginosa. This resistance to the affinity label is not due to a mutation within the primary structure of the enzyme. A single base change (C----A) located 87 bp upstream of the initiation codon for the gene for mandelate racemase was detected in three independent isolates of alpha-phenylglycidate-resistant colonies and appears responsible for a 30-fold increase in the amount of mandelate racemase encoded by the gene contained in the plasmid.(ABSTRACT TRUNCATED AT 250 WORDS)
Affinity labelling of pancreatic RNase with 4-(N-2-chloroethyl-N-methylamino)benzylamide and (N----P) N-methylimidazolide of d(pTpA) results in the formation of monomodified enzyme derivatives retaining partially enzymatic activity. These data together with some cases described in the literature are considered as suggesting the dynamic nature of the enzyme-reagent complex represented by a set of states differing in the probability of intra-complex reaction. In particular, modification may proceed in a low probability state with an especially favorable mutual orientation of reagent and some protein residue remote from the active site of the enzyme resulting in the removal of the covalently attached reagent moiety from the active center.
Opioid binding properties of Tyr-D-Ser-Gly-Phe-Leu-Thr-NH-NH-Gly-Mal (DSLET-Mal), a novel enkephalin-framed affinity label, was determined in rat brain membranes. In competition studies the ligand showed high affinity for the delta opioid sites, labelled by [(3)H][Ile(5,6)]deltorphin II (K(i) = 8 nM), whereas its binding to the mu ([(3)H]DAMGO) and kappa ([(3)H]EKC) sites was weaker. Preincubation of the rat brain membranes with DSLET-Mal at micromolar concentrations resulted in a wash-resistant and dose-dependent inhibition of the [(3)H][Ile(5,6)]deltorphin II binding sites (96% blocking at 10 microM concentration). Intracerebroventricular (ICV) administration of DSLET-Mal reduced the density of delta opioid receptors and had no effect on mu and kappa receptors, as determined by saturation binding studies. [Ile(5, 6)]deltorphin II-stimulated [(35)S]GTPgammaS binding was determined in membrane preparations of different brain areas of the ICV-treated animals. In both frontal cortex and hippocampus DSLET-Mal significantly decreased G protein activation by the delta agonist, having no effect on DAMGO stimulated [(35)S]GTPgammaS binding. DSLET-Mal had qualitatively similar effects on both receptor binding and G protein activation. These characteristics of the compound studied suggest that DSLET-Mal can serve as an affinity label for further studies of the delta-opioid receptors.
Brain sigma-type receptors and phencyclidine receptors are thought to mediate the psychotomimetic effects of benzomorphans and phencyclidine in humans. Recently, we reported the characterization of a selective sigma receptor ligand, 1,3-di-o-tolyl-guanidine (DTG), that shows negligible crossreactivity with phencyclidine receptors. Here we describe the synthesis and characterization of an isothiocyanate derivative of DTG, di-o-tolyl-guanidine-isothiocyanate (DIGIT). Guinea pig brain membranes treated with nanomolar doses of DIGIT followed by extensive washing exhibit a dose dependent reduction of [3H]DTG and (+)[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [+)[3H]3-PPP) binding to sigma receptors. Binding of radiolabelled ligands for phencyclidine, mu-opioid, benzodiazepine and dopamine-D2 receptors is not affected by membrane treatment with DIGIT, indicating specificity of the affinity label for sigma-type receptors. Treatment of DIGIT-derivatized membranes with 2 M NaCl does not result in recovery of sigma binding activity, suggesting that DIGIT's interaction with sigma receptors is not of an ionic nature. Equilibrium saturation binding experiments show that the inhibition of [3H]DTG binding to sigma receptors by DIGIT pretreatment of membranes is attributable to an irreversible reduction in the affinity (increase in Kd) of sigma receptors for DTG. The finding that sigma receptors are irreversibly modified by DIGIT whereas phencyclidine receptors are not affected suggests that sigma receptors are physically separate from phencyclidine receptors. The availability of a selective affinity label for the sigma receptor should facilitate the purification of the receptors and the characterization of sigma-type pharmacological effects in vivo and in vitro.
Preincubation of Escherichia coli DNA polymerase I (pol I) with 5'-fluorosulfonylbenzoyladenosine (5'-FSBA) results in an irreversible inactivation of DNA polymerase activity with concomitant covalent binding of 5'-FSBA to enzyme. pol I-associated 3'-5' exonuclease activity, however, remains unaffected. Kinetic studies of inactivation indicate that the degree of inactivation is directly proportional to the concentration of 5'-FSBA and increases linearly with time. The presence of the metal chelate form of dNTP substrates or template primer, but not the template or primer alone, protects the enzyme from inactivation by 5'-FSBA. A complete inactivation of polymerase activity occurs when 2 mol of 5'-FSBA are covalently linked to 1 mol of enzyme, suggesting two sites of modification. Tryptic peptide mapping of 5'-FSBA-treated enzyme revealed the presence of two distinct peptides containing the affinity label, confirming the presence of two reactive sites in the enzyme. However, we find that only one of the two sites is essential for the polymerase activity since, in the presence of substrate dNTP or template primer during preincubation of enzyme with 5'-FSBA, incorporation of the affinity label is reduced by only 1 mol. Peptide analysis of dNTP or template primer-protected enzyme further revealed that a peptide eluting at 35 min from the C-18 matrix was protected from the 5'-FSBA reaction. It is therefore concluded that this peptide contains the domain essential for polymerase activity. Staphylococcus aureus V-8 protease digestion, amino acid composition, and sequence analysis of this peptide revealed this domain to span residues 669 to 687 in the primary amino acid sequence of pol I, and arginine 682 was found to be the site of 5'-FSBA reactivity.
The formyl peptide chemotaxis receptor of rabbit neutrophils and purified rabbit neutrophil plasma membranes has been identified by several affinity labeling techniques: covalent affinity cross-linking of N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys (125I-hexapeptide) to the membrane-bound receptor with either dimethyl suberimidate or ethylene glycol bis(succinimidyl succinate) and photoactivation of N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-N epsilon-[6-[(4-azido-2-nitrophenyl)amino]hexanoyl]Lys(125I-PAL). These techniques specifically identify the receptor as a polypeptide that migrates as a broad band on sodium dodecyl sulfate-polyacrylamide electrophoresis, with Mr 50 000-65 000. The receptor has been solubilized in active form from rabbit neutrophil membranes with the detergents 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin and from whole cells with CHAPS. Chemotaxis receptor activity was measured by the ability of the solubilized membrane material to bind 125I-hexapeptide or fMet-Leu-[3H]Phe with gel filtration or rapid filtration through poly(ethylenimine)- (PEI) treated filters as assay systems. 125I-PAL was specifically cross-linked to the same molecular weight material in the CHAPS and digitonin solubilized extract, but no specific labeling of the receptor was seen when membranes were extracted with Nonidet P-40 and Triton X-100. Therefore, although a large number of detergents are able to solubilize the receptor, it appears that some release the receptor in an inactive form. The ligand binding characteristics of fMet-Leu-[3H]Phe to the CHAPS-solubilized receptor shared properties with the membrane-bound formyl peptide receptor, both of which showed curvilinear, concave-upward Scatchard plots. Computer curve fitting with NONLIN and statistical analyses of the binding data indicated that for both the membrane-bound and solubilized receptors a two saturable sites model fitted the data significantly better (p less than 0.01) than did a one saturable site model. The characteristics of the two saturable sites model for the soluble receptor were a high-affinity site with a KD value of 1.25 +/- 0.45 nM and a low-affinity site with a KD value of 19.77 +/- 3.28 nM. A total of 35% of the two sites detected was of the higher affinity. In addition, a Hill coefficient of 0.61 +/- 0.12 was observed.
The photoinduced reaction of phenylalanyl-tRNA synthetase (E.C.6.1.1.20) from E.coli MRE-600 with tRNAphe containing photoreative p-N3-C6H4-NHCOCH2-group attached to 4-thiouridine sU8 (azido-tRNAphe) was investigated. The attachment of this group does not influence the dissociation constant of the complex of Phe-tRNAphe with the enzyme, however it results in sevenfold increase of Km in the enzymatic aminoacylation of tRNAphe. Under irradiation at 300 nm at pH 5.8 the covalent binding of [14C]-Phe-azido-tRNAphe to the enzyme takes place 0.3 moles of the reagent being attached per mole of the enzyme. tRNA prevents the reaction. Phenylalanine, ATP,ADP,AMP, adenosine and pyrophosphate (2.5 xx 10(-3) M) don't affect neither the stability of the tRNA-enzyme complex nor the rate of the affinity labelling. The presence of the mixture of either phenylalanine or phenylalaninol with ATP as well as phenylalaninol adenylate exhibits 50% inhibition of the photoinduced reaction. Therefore, the reaction of [14C]-Phe-azido-tRNA with the enzyme is significantly less sensitive to the presence of the ligands than the reaction of chlorambucilyl-tRNA with the reactive group attached to the acceptor end of the tRNA studied in 1. It has been concluded that the kinetics of the affinity labelling does permit to discriminate the influence of the low molecular weight ligands of the enzyme on the different sites of the tRNA enzyme interaction.
RNA polymerase was treated in the presence of promoter-containing templates with 16 affinity reagents, derivatives on NMPs, NDPs and NTPs with reactive substituents at the terminal phosphate. This treatment was followed by addition of a pyrimidine [alpha-32P]NTP. Due to 'catalytic competence' of some of the residues of the affinity reagents bound covalently near the active center at the first stage, active-center-catalyzed synthesis of a phosphodiester bond occurred, and radioactive residues with the general formula -pNpN (where p = radioactive phosphate) appeared covalently attached to the enzyme. Such affinity labelling was super-selective because affinity reagent residues bound outside the active center were not elongated and thus remained non-radioactive. Labelling took place only when the combination of the reagent and [alpha-32P]NTP corresponded to the sequence of nucleotides of the promoter. With reagents having short 'arms', only the beta subunit was labelled; the targets were His and/or Lys residues. With reagents having longer 'arms', the sigma subunit was also labelled.
The effect of ribosomal antibiotics on the photoinduced affinity labeling of Escherichia coli ribosomes by puromycin [Cooperman, B.S., Jaynes, E.N., Brunswick, D.J., & Luddy, M.A. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1974; Jaynes, E.N. Jr., Grant, P.G., Giangrande, G., Wieder, R., & Cooperman, B.S. (1978) Biochemistry 17, 561] has been studied. Although blasticidin S, sparsomycin, lincomycin, and erythromycin are essentially without effect, major changes are seen on addition of either chloramphenicol or tetracycline. The products of photoincorporation have been characterized by one- and two-dimensional gel electrophoresis and by specific immunoprecipitation with antibodies to ribosomal proteins. In the presence of chloramphenicol, protein S14 becomes the major labeled protein. In the presence of tetracycline, L23 remains the major labeled protein, but the yield of labeled ribosomes is enormously increased, and the labeling is more specific for L23. These results are discussed in terms of the known modes of action of these antibiotics and the photoreactivity of tetracycline.
BACKGROUND: Many studies have reported apparent associations between platelet monoamine oxidase (MAO) activity and susceptibility to alcoholism and other psychiatric conditions. Alcohol-dependent individuals generally exhibit lower platelet MAO activity compared with controls, and on this basis, platelet MAO has been proposed as a potential genetic marker for predisposition to alcoholism. However, several lines of evidence also suggest that MAO activity is reduced in both the brain and platelets of smokers. Many alcohol-dependent individuals are also tobacco users, and few studies have attempted to dissociate the effect of alcohol and tobacco use on MAO activity. METHODS: Platelet MAO-B activity in 629 subjects recruited as part of the WHO/ISBRA Study of State and Trait Markers of Alcohol Use and Dependence was assayed by using a high throughput fluorescence assay. Platelet MAO-B protein concentrations were measured by analysis of immunoblots probed with a polyclonal antibody selective for MAO. Quantitative measurements of affinity labeling of platelet MAO were made by using the selective MAO-B catalytic site antagonist [3H]Ro 19-6327. RESULTS: Multiple regression analysis revealed that subjects' gender, cigarette smoking, lifetime alcohol dependence, and recruitment site each contributed independently to the variance in platelet MAO activity levels. Female subjects had significantly higher MAO activity levels than males, whereas heavy smokers had significantly lower MAO activity levels than nonsmokers. Immunoblot measurement of platelet MAO-B protein demonstrated that females had significantly higher MAO-B protein concentrations. Platelet MAO-B protein concentrations did not differ significantly between smokers and nonsmokers but were lower in subjects with a diagnosis of lifetime alcohol dependence (DSM-IV) compared with subjects who were never alcohol dependent. Measurements of affinity labeling by [3H]Ro 19-6327 of platelet MAO correlated significantly with MAO activity levels (i.e., the lower MAO-B activity in smokers was mirrored by lower levels of [3H]Ro 19-6327 binding). CONCLUSIONS: In this international population, gender, cigarette smoking, lifetime history of alcohol dependence, and recruitment site were associated with lower platelet MAO activity levels. Differences in MAO activity associated with gender and lifetime alcohol dependence can be attributed largely to differences in MAO-B protein concentration, whereas those associated with smoking behavior may be the result of binding of an inhibitor contained in cigarette smoke to platelet MAO-B at catalytic site of MAO.
Polyketides are a diverse class of natural products having important clinical properties, including antibiotic, immunosuppressive and anticancer activities. They are biosynthesized by polyketide synthases (PKSs), which are modular, multienzyme complexes that sequentially condense simple carboxylic acid derivatives. The final reaction in many PKSs involves thioesterase-catalyzed cyclization of linear chain elongation intermediates. As the substrate in PKSs is presented by a tethered acyl carrier protein, introduction of substrate by diffusion is problematic, and no substrate-bound type I PKS domain structure has been reported so far. We describe the chemical synthesis of polyketide-based affinity labels that covalently modify the active site serine of excised pikromycin thioesterase from Streptomyces venezuelae. Crystal structures reported here of the affinity label-pikromycin thioesterase adducts provide important mechanistic insights. These results suggest that affinity labels can be valuable tools for understanding the mechanisms of individual steps within multifunctional PKSs and for directing rational engineering of PKS domains for combinatorial biosynthesis.