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C Khosla

Publications and source records attributed to C Khosla.

At least 19 recordsLinked to original sources
PubMed2001

Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase: versatility from a unique substrate channel.

As the first structural elucidation of a modular polyketide synthase (PKS) domain, the crystal structure of the macrocycle-forming thioesterase (TE) domain from the 6-deoxyerythronolide B synthase (DEBS) was solved by a combination of multiple isomorphous replacement and multiwavelength anomalous dispersion and refined to an R factor of 24.1% to 2.8-A resolution. Its overall tertiary architecture belongs to the alpha/beta-hydrolase family, with two unusual features unprecedented in this family: a hydrophobic leucine-rich dimer interface and a substrate channel that passes through the entire protein. The active site triad, comprised of Asp-169, His-259, and Ser-142, is located in the middle of the substrate channel, suggesting the passage of the substrate through the protein. Modeling indicates that the active site can accommodate and orient the 6-deoxyerythronolide B precursor uniquely, while at the same time shielding the active site from external water and catalyzing cyclization by macrolactone formation. The geometry and organization of functional groups explain the observed substrate specificity of this TE and offer strategies for engineering macrocycle biosynthesis. Docking of a homology model of the upstream acyl carrier protein (ACP6) against the TE suggests that the 2-fold axis of the TE dimer may also be the axis of symmetry that determines the arrangement of domains in the entire DEBS. Sequence conservation suggests that all TEs from modular polyketide synthases have a similar fold, dimer 2-fold axis, and substrate channel geometry.

Amino Acid Sequence↗
PubMed2001

In vitro reconstitution and analysis of the chain initiating enzymes of the R1128 polyketide synthase.

Biosynthesis of the carbon chain backbone of the R1128 substances is believed to involve the activity of a ketosynthase/chain length factor (ZhuB/ZhuA), an additional ketosynthase (ZhuH), an acyl transferase (ZhuC), and two acyl carrier proteins (ACPs; ZhuG and ZhuN). A subset of these proteins initiate chain synthesis via decarboxylative condensation between an acetyl-, propionyl-, isobutyryl-, or butyryl-CoA derived primer unit and a malonyl-CoA derived extender unit to yield an acetoacetyl-, beta-ketopentanoyl-, 3-oxo-4-methylpentanoyl-, or beta-ketohexanoyl-ACP product, respectively. To investigate the precise roles of ZhuH, ZhuC, ZhuG, and ZhuN, each protein was expressed in Escherichia coli and purified to homogeneity. Although earlier reports had proposed that ZhuC and its homologues played a role in primer unit selection, direct in vitro analysis of ZhuC showed that it was in fact a malonyl-CoA:ACP malonyltransferase (MAT). The enzyme could catalyze malonyl transfer but not acetyl- or propionyl-transfer onto R1128 ACPs or onto ACPs from other biosynthetic pathways, suggesting that ZhuC has broad substrate specificity with respect to the holo-ACP substrate but is specific for malonyl-CoA. Thus, ZhuC supplies extender units to both the initiating and elongating ketosynthases from this pathway. To interrogate the primer unit specificity of ZhuH, the kinetics of beta-ketoacyl-ACP formation in the presence of various acyl-CoAs and malonyl-ZhuG were measured. Propionyl-CoA and isobutyryl-CoA were the two most preferred substrates of ZhuH, although acetyl-CoA and butyryl-CoA could also be accepted and elongated. This specificity is not only consistent with earlier reports demonstrating that R1128B and R1128C are the major products of the R1128 pathway in vivo, but is also in good agreement with the properties of the ZhuH substrate binding pocket, as deduced from a recently solved crystal structure of the enzyme. Finally, to investigate the molecular logic for the occurrence of not one but two ACP genes within the R1128 gene cluster, the inhibition of ZhuH-catalyzed formation of beta-ketopentanoyl-ACP was quantified in the presence of apo-ZhuG or apo-ZhuN. Both apo-proteins were comparable inhibitors of the ZhuH catalyzed reaction, suggesting that the corresponding apo-proteins can be used interchangeably during chain initiation. Together, these results provide direct biochemical insights into the mechanism of chain initiation of an unusual bacterial aromatic PKS.

Carrier Proteins↗
PubMed2001

Malonyl-CoA:ACP transacylase from Streptomyces coelicolor has two alternative catalytically active nucleophiles.

Fatty acids and polyketides are synthesized by mechanistically and evolutionarily related multienzyme systems. Their carbon chain backbones are synthesized via repeated decarboxylative condensations of alpha-carboxylated building blocks onto a growing acyl chain. These alpha-carboxylated building blocks are transferred from the corresponding coenzyme A thioesters onto the phosphopantetheine arm of an acyl carrier protein (ACP) by acyl transferases, which operate by a ping-pong mechanism involving an acyl-O-serine intermediate. In the course of our studies on the malonyl-CoA:ACP transacylase (MAT) from Streptomyces coelicolor, we observed that an active-site Ser (97) --> Ala mutant retains activity as well as the ability to be covalently labeled by (14)C malonyl-CoA. Here we demonstrate that an alternative, catalytically competent nucleophile exists in the active site of this enzyme. Next to the active-site serine is a histidine residue that is conserved in some, but not all acyl transferases. The H96A mutant is also active and can be labeled, but an H96A/S97A double mutant is inactive and cannot be labeled. The ability of H96 to form a malonyl-imidazole adduct was confirmed by proteolysis, followed by radio-HPLC and mass spectrometric analysis of the S97A mutant enzyme. Kinetic analysis revealed that the k(cat) of the S97A mutant was within 10-fold that of the wild-type enzyme, whereas the K(M)s of the two enzymes were comparable. Sequence comparison with the E. coli MAT (whose X-ray structure is known) led to the identification of H201 as the putative base in the serine-histidine catalytic dyad of the S. coelicolor enzyme. The absence of MAT activity in the H201A mutant and the detection of weak activity in the H201Q mutant was consistent with this proposal. The implications of this unexpected finding are discussed.

Acyl-Carrier Protein S-Malonyltransferase↗
PubMed2001

Assessing the balance between protein-protein interactions and enzyme-substrate interactions in the channeling of intermediates between polyketide synthase modules.

6-Deoxyerythronolide B synthase (DEBS) is the modular polyketide synthase (PKS) that catalyzes the biosynthesis of 6-deoxyerythronolide B (6-dEB), the aglycon precursor of the antibiotic erythromycin. The biosynthesis of 6-dEB exemplifies the extraordinary substrate- and stereo-selectivity of this family of multifunctional enzymes. Paradoxically, DEBS has been shown to be an attractive scaffold for combinatorial biosynthesis, indicating that its constituent modules are also very tolerant of unnatural substrates. By interrogating individual modules of DEBS with a panel of diketides activated as N-acetylcysteamine (NAC) thioesters, it was recently shown that individual modules have a marked ability to discriminate among certain diastereomeric diketides. However, since free NAC thioesters were used as substrates in these studies, the modules were primed by a diffusive process, which precluded involvement of the covalent, substrate-channeling mechanism by which enzyme-bound intermediates are directly transferred from one module to the next in a multimodular PKS. Recent evidence pointing to a pivotal role for protein-protein interactions in the substrate-channeling mechanism has prompted us to develop novel assays to reassess the steady-state kinetic parameters of individual DEBS modules when primed in a more "natural" channeling mode by the same panel of diketide substrates used earlier. Here we describe these assays and use them to quantify the kinetic benefit of linker-mediated substrate channeling in a modular PKS. This benefit can be substantial, especially for intrinsically poor substrates. Examples are presented where the k(cat) of a module for a given diketide substrate increases >100-fold when the substrate is presented to the module in a channeling mode as opposed to a diffusive mode. However, the substrate specificity profiles for individual modules are conserved regardless of the mode of presentation. By highlighting how substrate channeling can allow PKS modules to effectively accept and process intrinsically poor substrates, these studies provide a rational basis for examining the enormous untapped potential for combinatorial biosynthesis via module rearrangement.

Acyl Carrier Protein↗
PubMed2001

Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster.

Streptomyces lavendulae produces complestatin, a cyclic peptide natural product that antagonizes pharmacologically relevant protein-protein interactions including formation of the C4b,2b complex in the complement cascade and gp120-CD4 binding in the HIV life cycle. Complestatin, a member of the vancomycin group of natural products, consists of an alpha-ketoacyl hexapeptide backbone modified by oxidative phenolic couplings and halogenations. The entire complestatin biosynthetic and regulatory gene cluster spanning ca. 50 kb was cloned and sequenced. It consisted of 16 ORFs, encoding proteins homologous to nonribosomal peptide synthetases, cytochrome P450-related oxidases, ferredoxins, nonheme halogenases, four enzymes involved in 4-hydroxyphenylglycine (Hpg) biosynthesis, transcriptional regulators, and ABC transporters. The nonribosomal peptide synthetase consisted of a priming module, six extending modules, and a terminal thioesterase; their arrangement and domain content was entirely consistent with functions required for the biosynthesis of a heptapeptide or alpha-ketoacyl hexapeptide backbone. Two oxidase genes were proposed to be responsible for the construction of the unique aryl-ether-aryl-aryl linkage on the linear heptapeptide intermediate. Hpg, 3,5-dichloro-Hpg, and 3,5-dichloro-hydroxybenzoylformate are unusual building blocks that repesent five of the seven requisite monomers in the complestatin peptide. Heterologous expression and biochemical analysis of 4-hydroxyphenylglycine transaminon confirmed its role as an aminotransferase responsible for formation of all three precursors. The close similarity but functional divergence between complestatin and chloroeremomycin biosynthetic genes also presents a unique opportunity for the construction of hybrid vancomycin-type antibiotics.

Base Sequence↗
PubMed2001

The loading module of rifamycin synthetase is an adenylation-thiolation didomain with substrate tolerance for substituted benzoates.

The rifamycin synthetase is primed with a 3-amino-5-hydroxybenzoate starter unit by a loading module that contains domains homologous to the adenylation and thiolation domains of nonribosomal peptide synthetases. Adenylation and thiolation activities of the loading module were reconstituted in vitro and shown to be independent of coenzyme A, countering literature proposals that the loading module is a coenzyme A ligase. Kinetic parameters for covalent arylation of the loading module were measured directly for the unnatural substrates benzoate and 3-hydroxybenzoate. This analysis was extended through competition experiments to determine the relative rates of incorporation of a series of substituted benzoates. Our results show that the loading module can accept a variety of substituted benzoates, although it exhibits a preference for the 3-, 5-, and 3,5-disubstituted benzoates that most closely resemble its biological substrate. The considerable substrate tolerance of the loading module of rifamycin synthetase suggests that the module has potential as a tool for generating substituted derivatives of natural products.

Actinomycetales↗
PubMed2001

Precursor-directed biosynthesis of 16-membered macrolides by the erythromycin polyketide synthase.

Streptomyces coelicolor CH999/pJRJ2 harbors a plasmid encoding DEBS(KS1 degrees ), a mutant form of 6-deoxyerythronolide B synthase that is blocked in the formation of 6-deoxyerythronolide B (1, 6-dEB) due to a mutation in the active site of the ketosynthase (KS1) domain that normally catalyzes the first polyketide chain elongation step of 6-dEB biosynthesis. Administration of (2E,4S,5R)-2,4-dimethyl-5-hydroxy-2-heptenoic acid, N-acetylcysteamine thioester (6) an unsaturated triketide analogue of the natural triketide chain elongation intermediate to cultures of S. coelicolor CH999/pJRJ2 results in formation of a 16-membered macrolactone, which is isolated in the hemiketal form 33. The formation of the octaketide 33 indicates that the triketide substrate has been processed by DEBS module 2 as if it were a diketide analogue. The substrate specificity of this novel reaction has been explored by the incubation of three additional analogues of the unsaturated triketide 6, compounds 18, 31, and 32, with S. coelicolor CH999/pJRJ2, resulting in the formation of the corresponding macrolactones 34, 35, and 36. By contrast, the unsaturated triketide 10, lacking a methyl group at C-2, did not give rise to any detectable macrolactone product when incubated with S. coelicolor CH999/pJRJ2.

Anti-Bacterial Agents↗
PubMed2001

Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli.

The macrocyclic core of the antibiotic erythromycin, 6-deoxyerythronolide B (6dEB), is a complex natural product synthesized by the soil bacterium Saccharopolyspora erythraea through the action of a multifunctional polyketide synthase (PKS). The engineering potential of modular PKSs is hampered by the limited capabilities for molecular biological manipulation of organisms (principally actinomycetes) in which complex polyketides have thus far been produced. To address this problem, a derivative of Escherichia coli has been genetically engineered. The resulting cellular catalyst converts exogenous propionate into 6dEB with a specific productivity that compares well with a high-producing mutant of S. erythraea that has been incrementally enhanced over decades for the industrial production of erythromycin.

Acyl Coenzyme A↗
PubMed2001

Intermodular communication in polyketide synthases: comparing the role of protein-protein interactions to those in other multidomain proteins.

Although the role of protein-protein interactions in transducing signals within biological systems has been extensively explored, their relevance to the channeling of intermediates in metabolism is not widely appreciated. Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) are two related families of modular megasynthases that channel covalently bound intermediates from one active site to the next. Recent biochemical studies have highlighted the importance of protein-protein interactions in these chain transfer processes. The information available on this subject is reviewed, and its possible mechanistic implications are placed in context by comparisons with selected well-studied multicomponent protein systems.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed2001

Selective protein-protein interactions direct channeling of intermediates between polyketide synthase modules.

Polyketide synthases (PKSs) have represented fertile targets for rational manipulation via protein engineering ever since their modular architecture was first recognized. However, the mechanistic principles by which biosynthetic intermediates are sequentially channeled between modules remain poorly understood. Here we demonstrate the importance of complementarity in a remarkably simple, repetitive structural motif within these megasynthases that has been implicated to affect intermodular chain transfer [Gokhale, R. S., et al. (1999) Science 284, 482]. The C- and N-terminal ends of adjacent PKS polypeptides are capped by short peptides of 20-40 residues. Mismatched sequences abolish intermodular chain transfer without affecting the activity of individual modules, whereas matched sequences can facilitate the channeling of intermediates between ordinarily nonconsecutive modules. Thus, in addition to substrate-PKS interactions and domain-domain interactions, these short interpolypeptide sequences represent a third determinant of selective chain transfer that must be taken into consideration in the protein engineering of PKSs. Preliminary biophysical studies on synthetic peptide mimics of these linkers suggest that they may adopt coiled-coil conformations.

Amino Acid Sequence↗
PubMed2001

Structure-activity relationships within a family of selectively cytotoxic macrolide natural products.

[figure: see text] We describe a semi-synthetic deglycosylated derivative of apoptolidin that retains considerable activity against the mitochondrial ATPase but has greatly reduced cellular cytotoxicity. We also demonstrate that a related antifungal natural product, cytovaricin, inhibits the same molecular target. Structural comparison of these macrolides provides insights into their conserved features that are presumably important for biological activity and identifies promising avenues at the interface of organic synthesis and biosynthesis for the generation of new selective cytotoxic agents.

Anti-Bacterial Agents↗
PubMed2001

Modular enzymes.

Although modular macromolecular devices are encountered frequently in a variety of biological situations, their occurrence in biocatalysis has not been widely appreciated. Three general classes of modular biocatalysts can be identified: enzymes in which catalysis and substrate specificity are separable, multisubstrate enzymes in which binding sites for individual substrates are modular, and multienzyme systems that can catalyse programmable metabolic pathways. In the postgenomic era, the discovery of such systems can be expected to have a significant impact on the role of enzymes in synthetic and process chemistry.

Catalysis↗
PubMed2001

Process development and metabolic engineering for the overproduction of natural and unnatural polyketides.

Polyketide natural products are a rich source of bioactive substances that have found considerable use in human health and agriculture. Their complex structures require that they be produced via fermentation processes. This review describes the strategies and challenges used to develop practical fermentation strains and processes for polyketide production. Classical strain improvement procedures, process development methods, and metabolic engineering approaches are described. The elucidation of molecular mechanisms that underlie polyketide biosynthesis has played an important role in each of these areas over the past few years.

Biological Factors↗
PubMed2001

Apoptolidin, a selective cytotoxic agent, is an inhibitor of F0F1-ATPase.

BACKGROUND: Apoptolidin is a macrolide originally identified on the basis of its ability to selectively kill E1A and E1A/E1B19K transformed rat glial cells while not killing untransformed glial cells. The goal of this study was to identify the molecular target of this newly discovered natural product. RESULTS: Our approach to uncovering the mechanism of action of apoptolidin utilized a combination of molecular and cell-based pharmacological assays as well as structural comparisons between apoptolidin and other macrocyclic polyketides with known mechanism of action. Cell killing induced by apoptolidin was independent of p53 status, inhibited by BCL-2, and dependent on the action of caspase-9. PARP was completely cleaved in the presence of 1 microM apoptolidin within 6 h in a mouse lymphoma cell line. Together these results suggested that apoptolidin might target a mitochondrial protein. Structural comparisons between apoptolidin and other macrolides revealed significant similarity between the apoptolidin aglycone and oligomycin, a known inhibitor of mitochondrial F0F1-ATP synthase. The relevance of this similarity was established by demonstrating that apoptolidin is a potent inhibitor of the F0F1-ATPase activity in intact yeast mitochondria as well as Triton X-100-solubilized ATPase preparations. The K(i) for apoptolidin was 4-5 microM. The selectivity of apoptolidin in the NCI-60 cell line panel was found to correlate well with that of several known anti-fungal natural products that inhibit the eukaryotic mitochondrial F0F1-ATP synthase. SIGNIFICANCE: Although the anti-fungal activities of macrolide inhibitors of the mitochondrial F0F1-ATP synthase such as oligomycin, ossamycin and cytovaricin are well-documented, their unusual selectivity toward certain cell types is not widely appreciated. The recent discovery of apoptolidin, followed by the demonstration that it is an inhibitor of the mitochondrial F0F1-ATP synthase, highlights the potential relevance of these natural products as small molecules to modulate apoptotic pathways. The mechanistic basis for selective cytotoxicity of mitochondrial ATP synthase inhibitors is discussed.

Animals↗
PubMed2001

Biosynthesis of polyketides in heterologous hosts.

Polyketide natural products show great promise as medicinal agents. Typically the products of microbial secondary biosynthesis, polyketides are synthesized by an evolutionarily related but architecturally diverse family of multifunctional enzymes called polyketide synthases. A principal limitation for fundamental biochemical studies of these modular megasynthases, as well as for their applications in biotechnology, is the challenge associated with manipulating the natural microorganism that produces a polyketide of interest. To ameliorate this limitation, over the past decade several genetically amenable microbes have been developed as heterologous hosts for polyketide biosynthesis. Here we review the state of the art as well as the difficulties associated with heterologous polyketide production. In particular, we focus on two model hosts, Streptomyces coelicolor and Escherichia coli. Future directions for this relatively new but growing technological opportunity are also discussed.

Biotechnology↗