Biomedical subjects

D E Cane

Publications and source records attributed to D E Cane.

At least 19 recordsLinked to original sources

PubMed1998

Spontaneous priming of a downstream module in 6-deoxyerythronolide B synthase leads to polyketide biosynthesis.

Modular polyketide synthases such as 6-deoxyerythronolide B synthase (DEBS) catalyze the biosynthesis of structurally complex natural products by repetitive condensation of simple carboxylic acid monomers. The synthase can be divided into groups of domains, called "modules", each of which is responsible for one cycle of chain extension and processing. The modular nature of these enzymes suggests that the biosynthetic pathway might be rationally reprogrammed by manipulation of synthases at the domain level. Although, several examples of successful engineering of DEBS have been reported, a critical issue which has not been well-studied is the tolerance of "downstream" active sites to nonnatural substrates. Here, we report that the terminal modules of DEBS, which normally process highly functionalized intermediates, are competent to carry out their natural functions on smaller, more simple substrates. Expressed in the absence of other DEBS proteins, the DEBS3 protein, which normally carries out the final two extension cycles in the synthesis of 6-deoxyerythronolide B (6-dEB), is spontaneously primed with a C3 carboxylic acid. This substrate is then extended through two condensation cycles to form a triketide. Tolerance of the "shortened" intermediates in the biosynthesis of this triketide, in combination with results reported elsewhere [Jacobsen, J. R., Hutchinson, C. R., Cane, D. E., and Khosla, C. (1997) Science 277, 367-369], suggests that relaxed substrate specificity may be a common feature of modular polyketide synthases. Interestingly, priming of DEBS3 appears to proceed, not by acyltransfer from propionyl-CoA, but by decarboxylation of an enzyme-bound methylmalonyl extender unit. This is the second example of decarboxylative priming within DEBS [see also Pieper, R., Gokhale, R. S., Luo, G., Cane, D. E., and Khosla, C. (1997) Biochemistry 36, 1846-1851] and suggests that, in the absence of an acceptable primer (or transferred intermediate), decarboxylative priming of ketosynthase domains may be a general property of modular polyketide synthases.

Amino Acid Sequence↗

PubMed1998

Functional orientation of the acyltransferase domain in a module of the erythromycin polyketide synthase.

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex and medicinally important natural products. These large multienzymes are organized into a series of functional units known as modules. Each dimeric module contains two catalytically independent clusters of active sites homologous to those of vertebrate fatty acid synthases. Earlier studies have shown that modules consist of head-to-tail homodimers in which ketosynthase (KS) and acyl carrier protein (ACP) domains are contributed by opposite subunits to form a catalytic center. Here, we probe the functional topology of the acyltransferase (AT) domain which transfers the methylmalonyl moiety of methylmalonyl-CoA onto the phosphopantetheine arm of the ACP domain. Using a bimodular derivative of DEBS, the AT domain of module 2 (AT2) was inactivated by site-directed mutagenesis. Heterodimeric protein pairs were generated in vitro between the inactivated AT2 (AT2 degrees) polypeptide and an inactive KS1 (KS1 degrees) or KS2 (KS2 degrees) protein. Both of these hybrid proteins supported polyketide synthesis, suggesting that AT2 can perform its function from either subunit. The apparent catalytic rate constants for each of the two hybrid protein pairs, KS1 degrees/AT2 degrees and KS2 degrees/AT2 degrees, were identical, indicating that no significant kinetic preference exists for a particular AT2-ACP2 combination. These results suggest that the AT domain can be shared between the two clusters of active sites within the same dimeric module. Such a novel structural organization might provide a functional advantage for the efficient biosynthesis of polyketides.

Acyltransferases↗

PubMed1997

Crystal structure of pentalenene synthase: mechanistic insights on terpenoid cyclization reactions in biology.

The crystal structure of pentalenene synthase at 2.6 angstrom resolution reveals critical active site features responsible for the cyclization of farnesyl diphosphate into the tricyclic hydrocarbon pentalenene. Metal-triggered substrate ionization initiates catalysis, and the alpha-barrel active site serves as a template to channel and stabilize the conformations of reactive carbocation intermediates through a complex cyclization cascade. The core active site structure of the enzyme may be preserved among the greater family of terpenoid synthases, possibly implying divergence from a common ancestral synthase to satisfy biological requirements for increasingly diverse natural products.

Alkyl and Aryl Transferases↗

PubMed1997

Precursor-directed biosynthesis of erythromycin analogs by an engineered polyketide synthase.

A genetic block was introduced in the first condensation step of the polyketide biosynthetic pathway that leads to the formation of 6-deoxyerythronolide B (6-dEB), the macrocyclic precursor of erythromycin. Exogenous addition of designed synthetic molecules to small-scale cultures of this null mutant resulted in highly selective multimilligram production of unnatural polyketides, including aromatic and ring-expanded variants of 6-dEB. Unexpected incorporation patterns were observed, illustrating the catalytic versatility of modular polyketide synthases. Further processing of some of these scaffolds by postpolyketide enzymes of the erythromycin pathway resulted in the generation of novel antibacterials with in vitro potency comparable to that of their natural counterparts.

Amino Acid Substitution↗

PubMed1997

Pre-steady-state kinetic analysis of the trichodiene synthase reaction pathway.

The pre-steady-state kinetics of the trichodiene synthase reaction were investigated by rapid chemical quench methods. The single-turnover rate was found to be 3.5-3.8 s-1, a rate 40 times faster than the steady-state catalytic rate (kcat = 0.09 s-1) for trichodiene synthase-catalyzed conversion of farnesyl diphosphate (FPP) to trichodiene at 15 degrees C. In a multiturnover experiment, a burst phase (kb = 4.2 s-1) corresponding to the accumulation of trichodiene on the surface of the enzyme was followed by a slower, steady-state release of products (klin = 0.086 s-1) which corresponds to kcat. These results strongly suggest that the release of trichodiene from the enzyme active site is the rate-limiting step in the overall reaction, while the consumption of FPP is the step which limits chemical catalysis at the active site. Single-turnover experiments with trichodiene synthase mutant D101E, for which the steady-state rate constant kcat is 1/3 of that of wild type, revealed that the mutation actually depresses the rate of FPP consumption by a factor of 100. The deuterium isotope effect on the consumption of [1-2H,1,2-14C]FPP was found to be 1.11 +/- 0.06. Single turnover reactions of [1,2-14C]FPP catalyzed by trichodiene synthase were carried out at 4, 15, or 30 degrees C in an effort to provide direct observation of the proposed intermediate nerolidyl diphosphate (NPP). However, no NPP was detected, indicating that the conversion of NPP must be too fast to be observed within the detection limits of the assay. Taken together, these observations suggest that the isomerization of FPP to NPP is the step which limits the rate of chemical catalysis in the trichodiene synthase reaction pathway.

Binding Sites↗

PubMed1997

Purification and characterization of bimodular and trimodular derivatives of the erythromycin polyketide synthase.

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex and medicinally important natural products. DEBS is a dimeric protein complex that consists of three large multidomain polypeptide chains, DEBS 1, DEBS 2, and DEBS 3. In turn, each polypeptide includes two modules, where one module is responsible for a single round of condensation and associated reduction reactions. A hybrid protein comprised of the first two modules of DEBS fused to a thioesterase domain (DEBS 1 + TE) was purified to homogeneity in a fully active form (Kcat = 4.8 min-1). Synthesis of the anticipated triketide lactone required the presence of (2RS)-methylmalonyl-CoA and NADPH. When available, propionyl-CoA is the preferred source of primer units. However, in its absence the enzyme can derive primer units via decarboxylation of a methylmalonyl extender. The two subunits of an engineered trimodular derivative of DEBS, DEBS 1 and module 3 of DEBS 2 linked to the TE domain (module 3 + TE), were also individually purified and reconstituted to produce the expected tetraketide lactone in vitro (Kcat = 0.23 min-1). The considerably lower specific activity of this trimodular PKS relative to its bimodular counterpart presumably reflects inefficient association between DEBS 1 and module 3 + TE. As expected, module 3 + TE could be efficiently cross-linked as a homodimer. In contrast, no cross-links were detectable between modules 2 and 3, even though biosynthesis of the tetraketide requires transient interactions to occur between these two modules. Since module 3 only contains the minimal set of active sites required in a module (a ketosynthase, an acyltransferase, and an acyl carrier protein domain) and is the first active unimodular protein to be purified to homogeneity, it represents an attractive target for future biophysical and structural studies.

Cell-Free System↗

PubMed1997

Molecular recognition of diketide substrates by a beta-ketoacyl-acyl carrier protein synthase domain within a bimodular polyketide synthase.

BACKGROUND: Modular polyketide synthases (PKSs) are large multifunctional proteins that catalyze the biosynthesis of structurally complex bioactive products. The modular organization of PKSs has allowed the application of a combinatorial approach to the synthesis of novel polyketides via the manipulation of these biocatalysts at the genetic level. The inherent specificity of PKSs for their natural substrates, however, may place limits on the spectrum of molecular diversity that can be achieved in polyketide products. With the aim of further understanding PKS specificity, as a route to exploiting PKSs in combinatorial synthesis, we chose to examine the substrate specificity of a single intact domain within a bimodular PKS to investigate its capacity to utilize unnatural substrates. RESULTS: We used a blocked mutant of a bimodular PKS in which formation of the triketide product could occur only via uptake and processing of a synthetic diketide intermediate. By introducing systematic changes in the native diketide structure, by means of the synthesis of unnatural diketide analogs, we have shown that the ketosynthase domain of module 2 (KS2 domain) in 6-deoxyerythronolide B synthase (DEBS) tolerates a broad range of variations in substrate structure, but it strongly discriminates against some others. CONCLUSIONS: Defining the boundaries of substrate recognition within PKS domains is crucial to the rationally engineered biosynthesis of novel polyketide products, many of which could be prepared only with great difficulty, if at all, by direct chemical synthesis or semi-synthesis. Our results suggest that the KS2 domain of DEBS1 has a relatively relaxed specificity that can be exploited for the design and synthesis of medicinally important polyketide products.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

PubMed1996

High level expression of Ricinus communis casbene synthase in Escherichia coli and characterization of the recombinant enzyme.

Casbene synthase catalyzes the cyclization of geranylgeranyl diphosphate (2) to casbene (1), a diterpene phytoalexin with antibacterial and antifungal activity that is produced by seedlings of castor bean (Ricinus communis L.) in response to fungal attack. We report the high-level expression of casbene synthase cDNA in Escherichia coli as insoluble inclusion bodies, the solubilization and refolding of active casbene synthase, and the kinetic and product analysis of the recombinant enzyme. To overcome problems apparently associated with the presence in the casbene synthase gene of rare Arg codons, as well as the intrinsic antibacterial activity of casbene itself, the casbene synthase gene was expressed in an E. coli host harboring the pSM102 vector that encodes the dnaY gene for tArg(AGA/G), using an expression vector, pET-21d(+), carrying the tightly controlled T7lac promoter.

Ricinus communis↗

PubMed1996

6-deoxyerythronolide B synthase 1 is specifically acylated by a diketide intermediate at the beta-ketoacyl-acyl carrier protein synthase domain of module 2.

We have used 6-deoxyerythronolide B synthase (DEBS) as a model system to investigate molecular recognition by a modular polyketide synthase (PKS). DEBS consists of three proteins (DEBS1, -2, and -3) that biosynthesize the polyketide skeleton of the antibiotic erythromycin from propionyl-CoA and methylmalonyl-CoA. Active sites within these multifunctional proteins are organized into biosynthetic "modules", each of which catalyzes a discrete round of polyketide chain elongation and adjusts the appropriate level of beta-ketoacylthioester reduction. Using DEBS1, we demonstrate that there is a substantial degree of molecular recognition in the processing of the natural diketide chain elongation intermediate. Exogenously added (2S,3R)-2-methyl-3-hydroxypentanoic acid N-acetylcysteamine thioester is exclusively recognized by its cognate beta-ketoacyl-acyl carrier protein synthase domain in module 2 (KS2). Labeled diketide specifically acylated DEBS1 in crude protein extracts and limited proteolysis localized the binding to module 2. The precise site of acylation in DEBS1 was established by the finding that a Cys2200 Ala mutant of DEBS1, lacking the KS2 active-site cysteine, did not undergo acylation by the diketide. Pretreatment of the wild-type protein with the beta-ketoacyl-ACP synthase inhibitor cerulenin also blocked acylation. These results indicate that in addition to the purely organizational consequences resulting from the order of active-site domains, the programming of polyketide biosynthesis by modular PKSs involves a substantial level of molecular recognition. This conclusion has important implications for the use of PKSs to rationally design novel polyketides.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

PubMed1996

Trichodiene synthase. Probing the role of the highly conserved aspartate-rich region by site-directed mutagenesis.

Trichodiene synthase catalyzes the cyclization of farnesyl diphosphate to the sesquiterpene hydrocarbon trichodiene. The enzyme normally requires a divalent cation, Mg2+, which can be substituted by Mn2+. Trichodiene synthase from Fusarium sporotrichioides has a highly conserved aspartate rich region, aa 100-104 (DDSKD). Three mutants were constructed by site-directed mutagenesis in which each aspartate residue was individually replaced by glutamate. The mutants were each overexpressed and purified to homogeneity. The importance of Asp100 and Asp101 for catalysis was established by the observation of an increase in Km as well as a reduction in kcat in the corresponding Glu mutants. Replacement of the Asp104 residue with Glu had little effect on either Km or kcat. All three mutants produced anomalous sesquiterpene products in addition to trichodiene when incubated with farnesyl diphosphate. Interestingly, when Mg2+ was replaced by Mn2+ in the incubation buffer, the kcat/Km of both wild type trichodiene synthase and the D104E dropped significantly, while those of the other two mutants were not much affected. The proportion of anomalous products increased significantly when the D100E and D101E mutants were incubated in the presence of Mn2+. These observations all lend weight to the proposal that the aspartate residues mediate substrate binding by chelation of the divalent metal ion. Asp100 and Asp101 appear to play a relatively more important role than Asp104.

Aspartic Acid↗

PubMed1996

Evidence for two catalytically independent clusters of active sites in a functional modular polyketide synthase.

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex and medicinally important natural products. These large multifunctional enzymes are organized into "modules", where each module contains active sites homologous to those of higher eucaryotic fatty acid synthases (FASs). Like FASs, modular PKSs are known to be dimers. Here we provide functional evidence for the existence of two catalytically independent clusters of active sites within a modular PKS. In three bimodular derivatives of DEBS, the ketosynthase domain of module 1 (KS-1) or module 2 (KS-2) or the acyl carrier protein domain of module 2 (ACP-2) was inactivated via site-directed mutagenesis. As expected, the purified proteins were unable to catalyze polyketide synthesis (although the KS-1 mutant could convert a diketide thioester into the predicted triketide lactone). Remarkably however, the KS-1/KS-2 and the KS-2/ACP-2 mutant pairs could efficiently complement each other and catalyze polyketide formation. In contrast, the KS-1 and ACP-2 mutants did not complement each other. On the basis of these and other results, a model is proposed in which the individual modules of a PKS dimer form head-to-tail homodimers, thereby generating two equivalent and independent clusters of active sites for polyketide biosynthesis. Specifically, each subunit contributes half of the KS and ACP domains in each cluster. A similar complementation approach should also be useful in dissecting the organization of the remaining types of active sites within this family of multienzyme assemblies. Finally, blocked systems, such as the KS-1 mutant described here, present a new strategy for the noncompetitive conversion of unnatural substrates into polyketides by modular PKSs.

Acyl Carrier Protein↗

PubMed1996

Erythromycin biosynthesis: exploiting the catalytic versatility of the modular polyketide synthase.

DEBS 1 + TE is a recombinant modular polyketide synthase (PKS) in which the first two biosynthetic modules of the 6-deoxyerythronolide B synthase are linked to the thioesterase domain normally found at the C-terminus of DEBS 3. Incubation of DEBS 1 + TE with propionyl-CoA, methylamalonyl-CoA, and NADPH gives the triketide lactone (2R,3S,4S,5R)-2,4-dimethyl-3, 5-dihydroxy-n-heptanoic acid delta-lactone (2), the cyclized form of the normal triketide chain elongation product of DEBS 1. In order to probe the molecular recognition features of the PKS and to explore its synthetic versatility, [2,3-13C2]-(2S,3R)-2-methyl-3-hydroxypentanoyl-NAC thioester (3), an analogue of the normal diketide chain elongation intermediate, and (2RS)-methyl-malonyl-CoA were incubated with DEBS 1 + TE, leading to the formation of the predicted labeled triketide ketolactone [4,5-13C2]-8, as established by 13C NMR analysis and comparison with spectra of synthetic 8. This stereoselective conversion illustrates the potential of using modular PKSs as multifunctional catalysts for the enzymatic synthesis of novel polyketides.

Erythromycin↗

PubMed1996

A functional chimeric modular polyketide synthase generated via domain replacement.

BACKGROUND: Modular polyketide synthases (PKSs), such as 6-deoxyerythronolide B synthase (DEBS), are large multifunctional enzymes that catalyze the biosynthesis of structurally complex and medically important natural products. Active sites within these assemblies are organized into 'modules', such that each module catalyzes the stereospecific addition of a new monomer onto a growing polyketide chain and also sets the reduction level of the beta-carbon atom of the resulting intermediate. The core of each module is made up of a 'reductive segment', which includes all, some, or none of a set of ketoreductase (KR), dehydratase, and enoylreductase domains, in addition to a large interdomain region which lacks overt function but may contribute to structural stability and inter-domain dynamics within modules. The highly conserved organization of reductive segments within modules suggests that they might be able to function in unnatural contexts to generate novel organic molecules. RESULTS: To investigate domain substitution as a method for altering PKS function, a chimeric enzyme was engineered. Using a bimodular derivative of DEBS (DEBS1+TE), the reductive segment of module 2, which includes a functional KR, was replaced with its homolog from module 3 of DEBS, which contains a (naturally occurring) nonfunctional KR. A recombinant strain expressing the chimeric gene produced the predicted ketolactone with a yield (35 %) comparable to that of a control strain in which the KR2 domain was retained but mutationally inactivated. CONCLUSIONS: These results demonstrate considerable structural tolerance within an important segment found in virtually every PKS module. The domain boundaries defined here could be exploited for the construction of numerous loss-of-function and possibly even gain-of-function mutants within this remarkable family of multifunctional enzymes.

Multienzyme Complexes↗

PubMed1995

Cell-free synthesis of polyketides by recombinant erythromycin polyketide synthases.

Modular polyketide synthases (PKSs) are complex multi-enzyme proteins that catalyse the bacterial biosynthesis of many pharmaceutically useful polyketides. The PKSs are organized into a series of modules, each containing the active catalytic sites required for one step in the synthesis process. Here we report a method for cell-free enzymatic synthesis of 6-deoxyerythronolide B (6-dEB), the parent molecule of the antibiotic erythromycin A, using recombinant 6-deoxyerythronolide B synthase (DEBS), a modular PKS with at least 28 distinct active sites. We have also synthesized in vitro a triketide lactone by using a truncated mutant of DEBS. The availability of such cell-free synthetic routes will allow direct investigation of the structural and mechanistic basis for the unusual combination of high substrate specificity and tolerance to genetic reprogramming found in this enzyme family.

Amino Acid Sequence↗

PubMed1995

Trichodiene synthase. Substrate specificity and inhibition.

The substrate specificity of the sesquiterpene synthase trichodiene synthase was examined by determining the Vmax and Km parameters for the natural substrate, trans,trans-farnesyl diphosphate (1), its stereoisomer, cis,trans-farnesyl diphosphate, and the tertiary allylic isomer, (3R)-nerolidyl diphosphate (3), using both the native fungal and recombinant enzymes. A series of farnesyl diphosphate analogs, 15, 16, 20, 7, 8, and 9, was also tested as inhibitors of trichodiene synthase. 10-Fluorofarnesyl diphosphate (15) was the most effective competitive inhibitor, with a K1 of 16 nM compared to the Km for 1 of 87 nM, while the ether analog of farnesyl diphosphate, 8, an extremely potent inhibitor of squalene synthase, showed only modest inhibition of trichodiene synthase, with a K1/Km of 70.

Binding, Competitive↗

PubMed1995

Trichodiene synthase. Identification of active site residues by site-directed mutagenesis.

Derivatization of 5,5'-dithiobis(2-nitrobenzoic acid)-treated trichodiene synthase with [methyl-14C]methyl methanethiosulfonate and analysis of the derived tryptic peptides suggested the presence of two cysteine residues at the active site. The corresponding C146A and C190A mutants were constructed by site-directed mutagenesis. The C190A mutant displayed partial but significantly reduced activity, with a reduction in kcat/Km of 3000 compared to the wild-type trichodiene synthase, while the C146A mutant was essentially inactive. A hybrid trichodiene synthase, constructed from amino acids 1-309 of the Fusarium sporotrichioides enzyme and amino acids 310-383 of the Gibberella pulicaris cyclase, had steady state kinetic parameters nearly identical to those of the wild-type F. sporotrichioides enzyme. From this parent hybrid, a series of mutants was constructed by site-directed mutagenesis in which the amino acids in the base-rich region, 302-306 (DRRYR), were systematically modified. Three of these mutants were overexpressed and purified to homogeneity. The importance of Arg304 for catalysis was established by the observation that the R304K mutant showed a more than 25-fold increase in Km, as well as a 200-fold reduction in kcat. In addition, analysis of the incubation products of the R304K mutant by gas chromatography-mass spectrometry (GC-MS) indicated that farnesyl diphosphate was converted not only to trichodiene but to at least two additional C15H24 hydrocarbons, mle 204. Replacement of the Tyr305 residue of trichodiene synthase with Phe had little effect on kcat, while increasing the Km by a factor of ca. 7-8.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Overproduction and characterization of the erythromycin C-12 hydroxylase, EryK.

Hydroxylation of C-12 is one of the final steps in the biosynthesis of erythromycin A (ErA). A point of uncertainty in the erythromycin pathway has been whether the C-12 hydroxylase operates on each of two possible substrates, erythromycin B (ErB) and erythromycin D (ErD). Stassi et al. have cloned the gene, designated eryK, which encodes the P-450 monooxygenase responsible for erythromycin C-12 hydroxylation in Saccharopolyspora erythraea [Stassi, D., Donadio, S., Staver, M. J., & Katz, L. (1993) J. Bacteriol. 175, 182-189]. We report the overproduction of EryK in Escherichia coli as insoluble inclusion bodies; the solubilization, refolding, and reconstitution of active holo-EryK; and kinetic confirmation of a 1200-1900-fold preference of the enzyme for ErD over the alternative C-12 hydroxylase substrate ErB. Our results indicate that ErB is a shunt metabolite in the erythromycin biosynthetic pathway.

Amino Acid Sequence↗

PubMed1995

Crystallization and preliminary X-ray diffraction analysis of recombinant pentalenene synthase.

Recombinant pentalenene synthase, a 42.5-kDa sesquiterpene cyclase originally isolated from Streptomyces UC5319 and cloned in Escherichia coli, has been crystallized in space group P6(3) with unit cell dimensions a = b = 183.5 A and c = 56.5 A. Hexagonal prismatic crystals, approximately 0.2 x 0.2 x 0.3 mm, diffract to approximately 2.9 A resolution using monochromatic synchrotron radiation. From the universal (and achiral) building block, farnesyl pyrophosphate, pentalenene synthase catalyzes the formation of four stereocenters in the construction of the three fused five-membered rings of pentalenene; this novel sesquiterpene is a precursor to the pentalenolactone family of antibiotics.

Crystallization↗