The product of dpsC confers starter unit fidelity upon the daunorubicin polyketide synthase of Streptomyces sp. strain C5

The biosynthesis of daunorubicin and its precursors proceeds via the condensation of nine C-2 units derived from malonyl-CoA onto a propionyl starter moiety. The daunorubicin polyketide biosynthesis gene cluster of Streptomyces sp. strain C5 has two unique open reading frames, dpsC and dpsD, encoding, respectively, a fatty acid ketoacyl synthase (KAS) III homologue that is lacking an active-site cysteine and a proposed acyl-CoA:acyl carrier protein acyltransferase. The two genes are positioned directly downstream of dpsA and dpsB which encode the alpha and beta components of the type II KAS, respectively. Expression of the dpsABCDEFGdauGI genes in Streptomyces lividans resulted in the formation of aklanonic acid, the first stable chromophore of the daunorubicin biosynthesis pathway. Deletion of dpsC, but not dpsD, from this gene set resulted in the formation of desmethylaklanonic acid, derived from an acetyl-CoA starter unit, and aklanonic acid, derived from propionyl-CoA, in a 60:40 ratio. Thus, DpsC contributes to the selection of propionyl-CoA as the starter unit but does not alone dictate it. A dpsCD deletion mutant of Streptomyces sp. strain C5 (C5VR5) still produced daunorubicin but, more significantly, anthracycline and anthracyclinone derivatives resulting from the use of acetyl-CoA as an alternative starter moiety. Expression of dpsC, but not dpsD, in mutant C5VR5 restored the wild-type phenotype. Among the new compounds was the new biosynthesis product feudomycin D. These results suggest that in the absence of DpsC, the daunorubicin PKS complex behaves promiscuously, utilizing both acetyl-CoA (ca. 60% of the time) and propionyl-CoA (ca. 40%) as starter units. The fact that DpsC is not required for initiation with propionyl-CoA is significant, as the information must then lie in other components of the PKS complex. We propose to call DpsC the propionyl starter unit "fidelity factor." Copyright 2001 Academic Press.     

The Streptomyces peucetius dpsC Gene Determines the Choice of Starter Unit in Biosynthesis of the Daunorubicin Polyketide

The starter unit used in the biosynthesis of daunorubicin is propionyl coenzyme A (CoA) rather than acetyl-CoA, which is used in the production of most of the bacterial aromatic polyketides studied to date. In the daunorubicin biosynthesis gene cluster of Streptomyces peucetius, directly downstream of the genes encoding the beta-ketoacyl:acyl carrier protein synthase subunits, are two genes, dpsC and dpsD, encoding proteins that are believed to function as the starter unit-specifying enzymes. Recombinant strains containing plasmids carrying dpsC and dpsD, in addition to other daunorubicin polyketide synthase (PKS) genes, incorporate the correct starter unit into polyketides made by these genes, suggesting that, contrary to earlier reports, the enzymes encoded by dpsC and dpsD play a crucial role in starter unit specification. Additionally, the results of a cell-free synthesis of 21-carbon polyketides from propionyl-CoA and malonyl-CoA that used the protein extracts of recombinant strains carrying other daunorubicin PKS genes to which purified DpsC was added suggest that this enzyme has the primary role in starter unit discrimination for daunorubicin biosynthesis.     

Enzymatic assembly of epothilones: the EpoC subunit and reconstitution of the EpoA-ACP/B/C polyketide and nonribosomal peptide interfaces

The biosynthesis of epothilones, a family of hybrid polyketide (PK)/nonribosomal peptide (NRP) antitumor agents, provides an ideal system to study a hybrid PK/NRP natural product with significant biomedical value. Here the third enzyme involved in epothilone production, the five domain 195 kDa polyketide synthase (PKS) EpoC protein, has been expressed and purified from Escherichia coli. EpoC was combined with the first two enzymes of the epothilone biosynthesis pathway, the acyl carrier protein (ACP) domain of EpoA and EpoB, to reconstitute the early steps in epothilone biosynthesis. The acyltransferase (AT) domain of EpoC transfers the methylmalonyl moiety from methylmalonyl-CoA to the holo HS-acyl carrier protein (ACP) in an autoacylation reaction. The ketosynthase (KS) domain of EpoC decarboxylates the methylmalonyl-S-EpoC acyl enzyme to generate the carbon nucleophile that reacts with methylthiazolylcarboxyl-S-EpoB. The resulting condensation product can be reduced in the presence of NADPH by the ketoreductase (KR) domain of EpoC and then dehydrated by the dehydratase (DH) domain to produce the methylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediate that serves as the acyl donor for subsequent elongation of the epothilone chain. The acetyl-CoA donor can be replaced with propionyl-CoA, isobutyryl-CoA, and benzoyl-CoA and the acyl chains accepted by both EpoB and EpoC subunits to produce ethyl-, isopropyl-, and phenylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediates, suggesting that future combinatorial biosynthetic variations in epothilone assembly may be feasible. These results demonstrate in vitro reconstitution of both the PKS/NRPS interface (EpoA-ACP/B) and the NRPS/PKS interface (EpoB/C) in the assembly line for this antitumor natural product.     

Microbial production of odd-chain fatty acids

Odd-chain fatty acids (OcFAs) and their derivatives have attracted much attention due to their beneficial physiological effects and their potential to be alternatives to advanced fuels. However, cells naturally produce even-chain fatty acids (EcFAs) with negligible OcFAs. In the process of biosynthesis of fatty acids (FAs), the acetyl-CoA serves as the starter unit for EcFAs, and propionyl-CoA works as the starter unit for OcFAs. The lack of sufficient propionyl-CoA, the precursor, is usually regarded as the main restriction for large-scale bioproduction of OcFAs. In recent years, synthetic biology strategies have been used to modify several microorganisms to produce more propionyl-CoA that would enable an efficient biosynthesis of OcFAs. This review discusses several reported and potential metabolic pathways for propionyl-CoA biosynthesis, followed by advances in engineering several cell factories for OcFAs production. Finally, trends and challenges of synthetic biology driven OcFAs production are discussed.     

Enzymes involved in fatty acid and polyketide biosynthesis in Streptomyces glaucescens: role of FabH and FabD and their acyl carrier protein specificity

Malonyl acyl carrier protein (ACP) is used as an extender unit in each of the elongation steps catalyzed by the type II dissociated fatty acid synthase (FAS) and polyketide synthase (PKS) of Streptomyces glaucescens. Initiation of straight-chain fatty acid biosynthesis by the type II FAS involves a direct condensation of acetyl-CoA with this malonyl-ACP to generate a 3-ketobutyryl-ACP product and is catalyzed by FabH. In vitro experiments with a reconstituted type II PKS system in the absence of FabH have previously shown that the acetyl-ACP (generated by decarboxylation of malonyl-ACP), not acetyl-CoA, is used to initiate tetracenomycin C (TCM C) biosynthesis. We have shown that sgFabH activity is present in S. glaucescens fermentations during TCM C production, suggesting that it could contribute to initiation of TCM C biosynthesis in vivo. Isotope incorporation studies with [CD3]acetate and [13CD3]acetate demonstrated significant intact retention of three deuteriums into the starter unit of palmitate and complete washout of deuterium label into the starter unit of TCM C. These observations provide evidence that acetyl-CoA is not used directly as a starter unit for TCM C biosynthesis in vivo and argue against an involvement of FabH in this process. Consistent with this conclusion, assays of the purified recombinant sgFabH with acetyl-CoA demonstrated activity using malonyl-ACP generated from either FabC (the S. glaucescens FAS ACP) (k(cat) 42.2 min(-1), K(m) 4.5 +/- 0.3 microM) or AcpP (the E. coli FAS ACP) (k(cat) 7.5 min(-1), K(m) 6.3 +/- 0.3 microM) but not TcmM (the S. glaucescens PKS ACP). In contrast, the sgFabD which catalyzes conversion of malonyl-CoA to malonyl-ACP for fatty acid biosynthesis was shown to be active with TcmM (k(cat) 150 min(-1), K(m) 12.2 +/- 1.2 microM), AcpP (k(cat) 141 min(-1), K(m) 13.2 +/- 1.6 microM), and FabC (k(cat) 560 min(-1), K(m) 12.7 +/- 2.6 microM). This enzyme was shown to be present during TCM C production and could play a role in generating malonyl-ACP for both processes. Previous demonstrations that the purified PKS ACPs catalyze self-malonylation and that a FabD activity is not required for polyketide biosynthesis are shown to be an artifact of the expression and purification protocols. The relaxed ACP specificity of FabD and the lack of a clear alternative are consistent with a role of FabD in providing malonyl-ACP precursors for PKS as well as FAS processes. In contrast, the ACP specificity of FabH, isotope labeling studies, and a demonstrated alternative mechanism for initiation of the PKS process provide unequivocal evidence that FabH is involved only in the FAS process.     

Ketosynthases in the initiation and elongation modules of aromatic polyketide synthases have orthogonal acyl carrier protein specificity

Many bacterial aromatic polyketides are synthesized by type II polyketide synthases (PKSs) which minimally consist of a ketosynthase-chain length factor (KS-CLF) heterodimer, an acyl carrier protein (ACP), and a malonyl-CoA:ACP transacylase (MAT). This minimal PKS initiates polyketide biosynthesis by decarboxylation of malonyl-ACP, which is catalyzed by the KS-CLF complex and leads to incorporation of an acetate starter unit. In non-acetate-primed PKSs, such as the frenolicin (fren) PKS and the R1128 PKS, decarboxylative priming is suppressed in favor of chain initiation with alternative acyl groups. Elucidation of these unusual priming pathways could lead to the engineered biosynthesis of polyketides containing novel starter units. Unique to some non-acetate-primed PKSs is a second catalytic module comprised of a dedicated homodimeric KS, an additional ACP, and a MAT. This initiation module is responsible for starter-unit selection and catalysis of the first chain elongation step. To elucidate the protein-protein recognition features of this dissociated multimodular PKS system, we expressed and purified two priming and two elongation KSs, a set of six ACPs from diverse sources, and a MAT. In the presence of the MAT, each ACP was labeled with malonyl-CoA rapidly. In the presence of a KS-CLF and MAT, all ACPs from minimal PKSs supported polyketide synthesis at comparable rates (k(cat) between 0.17 and 0.37 min(-1)), whereas PKS activity was attenuated by at least 50-fold in the presence of an ACP from an initiation module. In contrast, the opposite specificity pattern was observed with priming KSs: while ACPs from initiation modules were good substrates, ACPs from minimal PKSs were significantly poorer substrates. Our results show that KS-CLF and KSIII recognize orthogonal sets of ACPs, and the additional ACP is indispensable for the incorporation of non-acetate primer units. Sequence alignments of the two classes of ACPs identified a tyrosine residue that is unique to priming ACPs. Site-directed mutagenesis of this amino acid in the initiation and elongation module ACPs of the R1128 PKS confirmed the importance of this residue in modulating interactions between KSs and ACPs. Our study provides new biochemical insights into unusual chain initiation mechanisms of bacterial aromatic PKSs.     

Propionyl Coenzyme A (Propionyl-CoA) Carboxylase in Haloferax mediterranei: Indispensability for Propionyl-CoA Assimilation and Impacts on Global Metabolism

Propionyl coenzyme A (propionyl-CoA) is an important intermediate during the biosynthesis and catabolism of intracellular carbon storage of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in haloarchaea. However, the haloarchaeal propionyl-CoA carboxylase (PCC) and its physiological significance remain unclear. In this study, we identified a PCC that catalyzed propionyl-CoA carboxylation with an acetyl-CoA carboxylation side activity in Haloferax mediterranei. Gene knockout/complementation demonstrated that the PCC enzyme consisted of a fusion protein of a biotin carboxylase and a biotin-carboxyl carrier protein (PccA [HFX_2490]), a carboxyltransferase component (PccB [HFX_2478]), and an essential small subunit (PccX [HFX_2479]). Knockout of pccBX led to an inability to utilize propionate and a higher intracellular propionyl-CoA level, indicating that the PCC enzyme is indispensable for propionyl-CoA utilization. Interestingly, H. mediterranei DBX (pccBX-deleted strain) displayed multiple phenotypic changes, including retarded cell growth, decreased glucose consumption, impaired PHBV biosynthesis, and wrinkled cells. A propionyl-CoA concentration equivalent to the concentration that accumulated in DBX cells was demonstrated to inhibit succinyl-CoA synthetase of the tricarboxylic acid cycle in vitro. Genome-wide microarray analysis showed that many genes for glycolysis, pyruvate oxidation, PHBV accumulation, electron transport, and stress responses were affected in DBX. This study not only identified the haloarchaeal PCC for the metabolism of propionyl-CoA, an important intermediate in haloarchaea, but also demonstrated that impaired propionyl-CoA metabolism affected global metabolism in H. mediterranei.     

Policing starter unit selection of the enterocin type II polyketide synthase by the type II thioesterase EncL

Enterocin is an atypical type II polyketide synthase (PKS) product from the marine actinomycete 'Streptomyces maritimus'. The enterocin biosynthesis gene cluster (enc) codes for proteins involved in the assembly and attachment of the rare benzoate primer that initiates polyketide assembly with the addition of seven malonate molecules and culminates in a Favorskii-like rearrangement of the linear poly-β-ketone to give its distinctive non-aromatic, caged core structure. Fundamental to enterocin biosynthesis, which utilizes a single acyl carrier protein (ACP), EncC, for both priming with benzoate and elongating with malonate, involves maintaining the correct balance of acyl-EncC substrates for efficient polyketide assembly. Here, we report the characterization of EncL as a type II thioesterase that functions to edit starter unit (mis)priming of EncC. We performed a series of in vivo mutational studies, heterologous expression experiments, in vitro reconstitution studies, and Fourier-transform mass spectrometry-monitored competitive enzyme assays that together support the proposed selective hydrolase activity of EncL toward misprimed acetyl-ACP over benzoyl-ACP to facilitate benzoyl priming of the enterocin PKS complex. While this system resembles the R1128 PKS that also utilizes an editing thioesterase (ZhuC) to purge acetate molecules from its initiation module ACP in favor of alkylacyl groups, the enterocin system is distinct in its usage of a single ACP for both priming and elongating reactions with different substrates.     

Expression and characterization of polyketide synthase module involved in the late step of cephabacin biosynthesis from Lysobacter lactamgenus

The cephabacins produced by Lysobacter lactamgenus are beta-lactam antibiotics composed of a cephem nucleus, an acetate residue, and an oligopeptide side chain. In order to understand the precise implication of the polyketide synthase (PKS) module in the biosynthesis of cephabacin, the genes for its core domains, beta-ketoacyl synthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), were amplified and cloned into the pET-32b(+) expression vector. The sfp gene encoding a protein that can modify apo-ACP to its active holo-form was also amplified. The recombinant KS, AT, apo-ACP, and Sfp overproduced in the form of His6-tagged fusion proteins in E. coli BL21(DE3) were purified by nickel-affinity chromatography. Formation of stable peptidyl-S-KS was observed by in vitro acylation of the KS domain with the substrate [L-Ala-L-Ala-LAla- L-3H-Arg] tetrapeptide-S-N-acetylcysteamine, which is the evidence for the selective recognition of tetrapeptide produced by nonribosomal peptide synthetase (NRPS) in the NRPS/ PKS hybrid. In order to confirm whether malonyl CoA is the extender unit for acetylation of the peptidyl moiety, the AT domain, ACP domain, and Sfp protein were treated with 14C-malonyl-CoA. The results clearly show that the AT domain is able to recognize the extender unit and decarboxylatively acetylated for the elongation of the tetrapeptide. However, the transfer of the activated acetyl group to the ACP domain was not observed, probably attributed to the improper capability of Sfp to activate apo-ACP to the holo-ACP form.     

Mechanisms of growth inhibition by propionate and restoration of the growth by sodium bicarbonate or acetate in Rhodopseudomonas sphaeroides S

Mechanisms of growth inhibition by propionate on the growth of Rhodopseudomonas sphaeroides were studied. Partially purified pyruvate dehydrogenase complex (PDC) from R. sphaeroides was inhibited by propionyl-CoA, one of the metabolic intermediates of propionate, while propionate itself did not inhibit the enzyme. This suggests that the inhibitor of the growth in vivo is not propionate but propionyl-CoA. The inhibition by propionyl-CoA was competitive with respect to coenzyme A concentration. The K1 value for propionyl-CoA was 0.84 mM. Addition of NaHCO3, which restored the growth of this bacterium in the presence of propionate, increased the rate of propionate incorporation by 1.7-fold and decreased the intracellular level of propionyl-CoA by half. These findings suggest that HCO3-ion lowers the level of propionyl-CoA by accelerating its carboxylation reaction, which is catalyzed by propionyl-CoA carboxylase. Effects of NaHCO3 and acetate on the growth restoration were also studied by the use of propionyl-CoA carboxylase-deficient mutants. NaHCO3 did not restore the growth of the mutants, indicating an essential role of propionyl-CoA carboxylase on the restoration of growth by NaHCO3 as suggested above. Addition of acetate restores the growth of the mutants in the presence of propionate. Acetate probably restores the growth by supplying acetyl-CoA.     

Characterization of the propionyl-CoA synthetase (PrpE) enzyme of Salmonella enterica: residue Lys592 is required for propionyl-AMP synthesis

The propionyl-CoA synthetase (PrpE) enzyme of Salmonella enterica catalyzes the first step of propionate catabolism, i.e., the activation of propionate to propionyl-CoA. The PrpE enzyme was purified, and its kinetic properties were determined. Evidence is presented that the conversion of propionate to propionyl-CoA proceeds via a propionyl-AMP intermediate. Kinetic experiments demonstrated that propionate was the preferred acyl substrate (kcat/Km = 1644 mM(-1) x s(-1)). Adenosine 5'-propyl phosphate was a potent inhibitor of the enzyme, and inhibition kinetics identified a Bi Uni Uni Bi Ping Pong mechanism for the reaction catalyzed by the PrpE enzyme. Site-directed mutagenesis was used to change the primary sequence of the wild-type protein at positions G245A, P247A, K248A, K248E, G249A, K592A, and K592E. Mutant PrpE proteins were purified, and the effects of the mutations on enzyme activity were investigated. Both PrpEK592 mutant proteins (K592A and K592E) failed to convert propionate to propionyl-CoA, and plasmids containing these alleles of prpE failed to restore growth on propionate of S. enterica carrying null prpE alleles on their chromosome. Both PrpEK592 mutant proteins converted propionyl-AMP to propionyl-CoA, suggesting residue K592 played no discernible role in thioester bond formation. To the best of our knowledge, these mutant proteins are the first acyl-CoA synthetases reported that are defective in adenylation activity.     

Tandemly duplicated acyl carrier proteins, which increase polyketide antibiotic production, can apparently function either in parallel or in series

Polyketide biosynthesis involves the addition of subunits commonly derived from malonate or methylmalonate to a starter unit such as acetate. Type I polyketide synthases are multifunctional polypeptides that contain one or more modules, each of which normally contains all the enzymatic domains for a single round of extension and modification of the polyketide backbone. Acyl carrier proteins (ACP(s)) hold the extender unit to which the starter or growing chain is added. Normally there is one ACP for each ketosynthase module. However, there are an increasing number of known examples of tandemly repeated ACP domains, whose function is as yet unknown. For the doublet and triplet ACP domains in the biosynthetic pathway for the antibiotic mupirocin from Pseudomonas fluorescens NCIMB10586 we have inactivated ACP domains by inframe deletion and amino acid substitution of the active site serine. By deletion analysis each individual ACP from a cluster can provide a basic but reduced activity for the pathway. In the doublet cluster, substitution analysis indicates that the pathway may follow two parallel routes, one via each of the ACPs, thus increasing overall pathway flow. In the triplet cluster, substitution in ACP5 blocked the pathway. Thus ACP5 appears to be arranged "in series" to ACP6 and ACP7. Thus although both the doublet and triplet clusters increase antibiotic production, the mechanisms by which they do this appear to be different and depend specifically on the biosynthetic stage involved. The function of some ACPs may be determined by their location in the protein rather than absolute enzymic activity.     

Site-specific mutagenesis and domain substitutions in the loading module of the nystatin polyketide synthase,and their effects on nystatin biosynthesis in Streptomyces noursei

The loading module for the nystatin polyketide synthase (PKS) in Streptomyces noursei is represented by the NysA protein composed of a ketosynthase (KS(S)), acyltransferase, dehydratase, and an acyl carrier protein. The absolute requirement of this protein for initiation of nystatin biosynthesis was demonstrated by the in-frame deletion of the nysA gene in S. noursei. The role of the NysA KS(S) domain, however, remained unclear, since no data on the significance of the "active site" serine (Ser-170) residue in the loading modules of type I PKSs were available. Site-specific mutagenesis of Ser-170 both in the wild-type NysA and in the hybrid loading module containing malonyl-specific acyltransferase domain from the extender module had no effect on nystatin biosynthesis. A second mutation (S413N) of the NysA KS(S) domain was discovered that completely abolished the ability of the hybrids to restore nystatin biosynthesis, presumably by affecting the ability of the resulting proteins to catalyze the required substrate decarboxylation. In contrast, NysA and its Ser-170 mutants bearing the same S413N mutation were able to restore nystatin production to significant levels, probably by using acetyl-CoA as a starter unit. Together, these data suggest that the KS(S) domain of NysA differs from the KS(Q) domains found in the loading modules of several PKS type I systems in that the active site residue is not significant for its activity.     

Evolutionary affiliations within the superfamily of ketosynthases reflect complex pathway associations

Abstract Type I polyketide synthases are known to produce a wide range of medically and industrially important polyketides. The ketosynthase (KS) domain is required for the condensation of an extender unit onto the growing polyketide chain during polyketide biosynthesis. KSs represent a superfamily of complex biosynthetic pathway-associated enzymes found in prokaryotes, fungi, and plants. Although themselves functionally conserved, KSs are involved in the production of a structurally diverse range of metabolites. Degenerate oligonucleotide primers, designed for the amplification of KS domains, amplified KS domains from a range of organisms including cyanobacterial and dinoflagellates. KS domains detected in dinoflagellate cultures appear to have been amplified from the less than 3-μm filtrate of the nonaxenic culture. Phylogenetic analysis of sequences obtained during this study enabled the specific identification of KS domains of hybrid or mixed polyketide synthase/peptide synthetase complexes, required for the condensation of an extender unit onto an amino acid starter unit. The primer sets described in this study were also used for the detection of novel KS domains directly from environmental samples. The ability to predict function based on primary molecular structure will be critical for future discovery and rational engineering of polyketides.     

Isolation and characterization of a non-reducing polyketide synthase gene from the lichen-forming fungus Usnea longissima

Usnea longissima has long been used as a traditional medicine in China, India, Turkey, Canada and Europe. This lichen can produce several bioactive compounds that primarily belong to the polyketide family. The enzymes responsible for the production of these compounds are the polyketide synthases, but the biosynthetic processes in lichens are still unclear. In this study, a cultured mycobiont of Usnea longissima was used to isolate and characterize a polyketide synthase gene (UlPKS1). Complete sequence information regarding UlPKS1 (6,468 bp) was obtained by screening a Fosmid genomic library using a 512-bp fragment corresponding to part of the ketosynthase (KS) domain. Sequence analysis of UlPKS1 suggested that it contained features of a non-reducing fungal type I PKS with a starter unit of ACP transacylase (SAT), ketosynthase (KS), product template (PT), acyl carrier protein (ACP) transacylase, acyltransferase (AT) and thioesterase (TE) domain, and had five intervening introns. The domain organization of UlPKS1 (SAT-KS-AT-PT-ACP-ACP-TE) was quite similar to that of aromatic PKSs, and phylogenetic analysis showed that UlPKS1 belonged to the clade of lichenized fungal non-reducing PKS. RT-PCR analyses revealed that the expression of UlPKS1 was down-regulated by glycine and high concentrations of sorbitol, inositol and fructose and up-regulated by sucrose and glucose. Here, we introduce a non-reducing PKS gene in the lichen-forming fungus U. longissima, with a domain structure similar to the structure of orsellinic acid synthase A (OrsA) which is required for orsellinic acid biosynthesis in Aspergillus nidulans.     

Propionate increases neuronal histone acetylation, but is metabolized oxidatively by glia. Relevance for propionic acidemia

In propionic acidemia, propionate acts as a metabolic toxin in liver cells by accumulating in mitochondria as propionyl-CoA and its derivative, methylcitrate, two tricarboxylic acid cycle inhibitors. Little is known about the cerebral metabolism of propionate, although clinical effects of propionic acidemia are largely neurological. We found that propionate was metabolized oxidatively by glia: [3-(14)C]propionate injected into mouse striatum or cortex, gave a specific activity of glutamine that was 5-6 times that of glutamate, indicating metabolism in cells that express glutamine synthetase, i.e., glia. Further, cultured cerebellar astrocytes metabolized [3-(14)C]propionate; cultured neurons did not. However, both cultured cerebellar neurons and astrocytes took up [3H]propionate, and propionate exposure increased histone acetylation in cultured neurons and astrocytes as well as in hippocampal CA3 pyramidal neurons of wake mice. The inability of neurons to metabolize propionate may be due to lack of mitochondrial propionyl-CoA synthetase activity or transport of propionyl residues into mitochondria, as cultured neurons expressed propionyl-CoA carboxylase, a mitochondrial matrix enzyme, and oxidized isoleucine, which becomes converted into propionyl-CoA intramitochondrially. The glial metabolism of propionate suggests astrocytic vulnerability in propionic acidemia when intramitochondrial propionyl-CoA may accumulate. Propionic acidemia may alter both neuronal and glial gene expression by affecting histone acetylation.     

Mechanistic analysis of a type II polyketide synthase. Role of conserved residues in the beta-ketoacyl synthase-chain length factor heterodimer

Type II polyketide synthases (PKSs) are a family of multienzyme systems that catalyze the biosynthesis of polyfunctional aromatic natural products such as actinorhodin, frenolicin, tetracenomycin, and doxorubicin. A central component in each of these systems is the beta-ketoacyl synthase-chain length factor (KS-CLF) heterodimer. In the presence of an acyl carrier protein (ACP) and a malonyl-CoA:ACP malonyl transferase (MAT), this enzyme synthesizes a polyketide chain of defined length from malonyl-CoA. We have investigated the role of the actinorhodin KS-CLF in priming, elongation, and termination of its octaketide product by subjecting the wild-type enzyme and selected mutants to assays that probe key steps in the overall catalytic cycle. Under conditions reflecting steady-state turnover of the PKS, a unique acyl-ACP intermediate is detected that carries a long, possibly full-length, acyl chain. This species cannot be synthesized by the C169S, H309A, K341A, and H346A mutants of the KS, all of which are blocked in early steps in the PKS catalytic cycle. These four residues are universally conserved in all known KSs. Malonyl-ACP alone is sufficient for kinetically and stoichiometrically efficient synthesis of polyketides by the wild-type KS-CLF, but not by heterodimers that carry the mutations listed above. Among these mutants, C169S is an efficient decarboxylase of malonyl-ACP, but the H309A, K341A, and H346A mutants are unable to catalyze decarboxylation. Transfer of label from [(14)C]malonyl-ACP to the nucleophile at position 169 in the KS can be detected for the wild-type enzyme and for the C169S and K341A mutants, but not for the H309A mutant and only very weakly for the H346A mutant. A model is proposed for decarboxylative priming and extension of a polyketide chain by the KS, where C169 and H346 form a catalytic dyad for acyl chain attachment, H309 positions the malonyl-ACP in the active site and supports carbanion formation by interacting with the thioester carbonyl, and K341 enhances the rate of malonyl-ACP decarboxylation via electrostatic interaction. Our data also suggest that the ACP and the KS dissociate after each C-C bond forming event, and that the newly extended acyl chain is transferred back from the ACP pantetheine to the KS cysteine before dissociation can occur. Chain termination is most likely the rate-limiting step in polyketide biosynthesis. Within the act CLF, neither the universally conserved S145 residue nor Q171, which aligns with the active site cysteine of the ketosynthase, is essential for PKS activity. The results described here provide a basis for a better understanding of the catalytic cycle of type II PKSs and fatty acid synthases.     

Comparative metabolism of branched-chain amino acids to precursors of juvenile hormone biogenesis in corpora allata of lepidopterous versus nonlepidopterous insects

Comparative studies were performed on the role of branched-chain amino acids (BCAA) in juvenile hormone (JH) biosynthesis using several lepidopterous and nonlepidopterous insects. Corpora cardiaca-corpora allata complexes (CC-CA, the corpora allata being the organ of JH biogenesis) were maintained in culture medium containing a uniformly 14C-labeled BCAA, together with [methyl-3H]methionine as mass marker for JH quantification. BCAA catabolism was quantified by directly analyzing the medium for the presence of 14C-labeled propionate and/or acetate, while JHs were extracted, purified by liquid chromatography, and subjected to double-label liquid scintillation counting. Our results indicate that active BCAA catabolism occurs within the CC-CA of lepidopterans, and this efficiently provides propionyl-CoA (from isoleucine or valine) for the biosynthesis of the ethyl branches of JH I and II. Acetyl-CoA, formed from isoleucine or leucine catabolism, is also utilized by lepidopteran CC-CA for biosynthesizing JH III and the acetate-derived portions of the ethyl-branched JHs. In contrast, CC-CA of nonlepidopterans fail to catabolize BCAA. Consequently, exogenous isoleucine or leucine does not serve as a carbon source for the biosynthesis of JH III by these glands, and no propionyl-CoA is produced for genesis of ethyl-branched JHs. This is the first observation of a tissue-specific metabolic difference which in part explains why these novel homosesquiterpenoids exist in lepidopterans, but not in nonlepidopterans.     

Reconstituting modular activity from separated domains of 6-deoxyerythronolide B synthase

The hallmark of a type I polyketide synthase (PKS), such as the 6-deoxyerythronolide B synthase (DEBS), is the presence of catalytic modules comprised of covalently fused domains acting together to catalyze one round of chain elongation. In addition to an obligate ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP), a module may also include a ketoreductase (KR), dehydratase (DH), and/or enoyl reductase (ER) domain. The size, flexibility, and fixed domain-domain stoichiometry of these PKS modules present challenges for structural, mechanistic, and protein-engineering studies. Here, we have harnessed the power of limited proteolysis and heterologous protein expression to isolate and characterize individual domains of module 3 of DEBS, a 150-kD protein consisting of a KS, an AT, an ACP, and an inactive KR domain. Two interdomain boundaries were identified via limited proteolysis, which led to the production of a 90-kD KS-AT, a 142-kD KS-AT-KR(0), and a 10-kD ACP as structurally stable stand-alone proteins. Each protein was shown to possess the requisite catalytic properties. In the presence of the ACP, both the KS-AT and the KS-AT-KR(0) proteins were able to catalyze chain elongation as well as the intact parent module. Separation of the KS from the ACP enabled direct interrogation of the KS specificity for both the nucleophilic substrate and the partner ACP. Malonyl and methylmalonyl extender units were found to be equivalent substrates for chain elongation. Whereas ACP2 and ACP4 of DEBS could be exchanged for ACP3, ACP6 was a substantially poorer partner for the KS. Remarkably, the newly identified proteolytic sites were conserved in many PKS modules, raising the prospect of developing improved methods for the construction of hybrid PKS modules by engineering domain fusions at these interdomain junctions.