Probing Reactivity and Substrate Specificity of Both Subunits of the Dimeric \u3ci\u3eMycobacterium tuberculosis\u3c/i\u3e FabH Using alkyl-CoA Disulfide Inhibitors and acyl-CoA Substrates

The dimeric Mycobacterium tuberculosis FabH (mtFabH) catalyses a Claisen-type condensation between an acyl-CoA and malonyl-acyl carrier protein (ACP) to initiate the Type II fatty acid synthase cycle. To analyze the initial covalent acylation of mtFabH with acyl-CoA, we challenged it with mixture of C6-C20 acyl-CoAs and the ESI-MS analysis showed reaction at both subunits and a strict specificity for C12 acyl CoA. Crystallographic and ESI-MS studies of mtFabH with a decyl-CoA disulfide inhibitor revealed a decyl chain bound in acyl-binding channels of both subunits through disulfide linkage to the active site cysteine. These data provide the first unequivocal evidence that both subunits of mtFabH can react with substrates or inhibitor. The discrepancy between the observed C12 acyl-CoA substrate specificity in the initial acylation step and the higher catalytic efficiency of mtFabH for C18-C20 acyl-CoA substrates in the overall mtFabH catalyzed reaction suggests a role for M. tuberculosis ACP as a specificity determinant in this reaction

Separate Entrance and Exit Portals for Ligand Traffic in Mycobacterium tuberculosis FabH

SummaryMycobacterium tuberculosis FabH initiates type II fatty acid synthase-catalyzed formation of the long chain (C16–C22) acyl-coenzyme A (CoA) precursors of mycolic acids, which are major constituents of the bacterial cell envelope. Crystal structures of M. tuberculosis FabH (mtFabH) show the substrate binding site to be a buried, extended L-shaped channel with only a single solvent access portal. Entrance of an acyl-CoA substrate through the solvent portal would require energetically unfavorable reptational threading of the substrate to its reactive position. Using a class of FabH inhibitors, we have tested an alternative hypothesis that FabH exists in an “open” form during substrate binding and product release, and a “closed” form in which catalysis and intermediate steps occur. This hypothesis is supported by mass spectrometric analysis of the product profile and crystal structures of complexes of mtFabH with these inhibitors

Alkyl-CoA Disulfides as Inhibitors and Mechanistic Probes for FabH Enzymes

SummaryThe first step of the reaction catalyzed by the homodimeric FabH from a dissociated fatty acid synthase is acyl transfer from acyl-CoA to an active site cysteine. We report that C1 to C10 alkyl-CoA disulfides irreversibly inhibit Escherichia coli FabH (ecFabH) and Mycobacterium tuberculosis FabH with relative efficiencies that reflect these enzymes' differential acyl-group specificity. Crystallographic and kinetic studies with MeSSCoA show rapid inhibition of one monomer of ecFabH through formation of a methyl disulfide conjugate with this cysteine. Reaction of the second subunit with either MeSSCoA or acetyl-CoA is much slower. In the presence of malonyl-ACP, the acylation rate of the second subunit is restored to that of the native ecFabH. These observations suggest a catalytic model in which a structurally disordered apo-ecFabH dimer orders on binding either the first substrate, acetyl-CoA, or the inhibitor MeSSCoA, and is restored to a disordered state on binding of malonyl-ACP

Peripherally restricted transthyretin-based delivery system for probes and therapeutics avoiding opioid-related side effects

Several investigations into the sites of action of opioid analgesics have utilized peripherally acting mu-opioid receptor antagonists (PAMORAs), which have been incorrectly assumed to possess limited permeability across the blood-brain barrier. Unfortunately, the poor pharmacokinetic properties of current PAMORAs have resulted in misunderstandings of the role of central nervous system and gastrointestinal tract in precipitating side effects such as opioid-induced constipation. Here, we develop a drug delivery approach for restricting the passage of small molecules across the blood-brain barrier. This allows us to develop naloxone- and oxycodone-based conjugates that display superior potency, peripheral selectivity, pharmacokinetics, and efficacy in rats compared to other clinically used PAMORAs. These probes allow us to demonstrate that the mu-opioid receptors in the central nervous system have a fundamental role in precipitating opioid-induced constipation. Therefore, our conjugates have immediate use as pharmacological probes and potential therapeutic agents for treating constipation and other opioid-related side effects

Identification of the Tirandamycin Biosynthetic Gene Cluster from Streptomyces sp. 307-9

The structurally intriguing bicyclic ketal moiety of tirandamycin is common to several acyl-tetramic acid antibiotics, and is a key determinant of biological activity. We have identified the tirandamycin biosynthetic gene cluster from the environmental marine isolate Streptomyces sp. 307–9, thus providing the first genetic insight into the biosynthesis of this natural product scaffold. Sequence analysis revealed a hybrid polyketide synthase–nonribosomal peptide synthetase gene cluster with a colinear domain organization, which is entirely consistent with the core structure of the tirandamycins. We also identified genes within the cluster that encode candidate tailoring enzymes for elaboration and modification of the bicyclic ketal system. Disruption of tamI , which encodes a presumed cytochrome P450, led to a mutant strain deficient in production of late stage tirandamycins that instead accumulated tirandamycin C, an intermediate devoid of any post assembly-line oxidative modifications.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/69167/1/cbic_200900658_sm_miscellaneous_information.pd

Empowering Therapeutic Peptides by Enhancing its in vivo Half-Life

The tremendous therapeutic potential of peptides has not been fulfilled and potential peptide therapies that have failed far outnumber the successes so far. A major challenge impeding the more widespread use of peptides as therapeutics is their poor pharmaco­kinetic profile, due to short in vivo half-life. Therefore, ex­tending the in vivo half-life of peptides is clearly desirable in order for their therapeutic potential to be realized, without the need for high doses and frequent administration. Our group is developing a novel strategy that will enhance the pharmacokinetic properties of thera­peutic peptides, which could decrease dosing frequency and im­prove patient convenience and compliance

Empowering Therapeutic Peptides by Enhancing its in vivo Half-Life

The tremendous therapeutic potential of peptides has not been fulfilled and potential peptide therapies that have failed far outnumber the successes so far. A major challenge impeding the more widespread use of peptides as therapeutics is their poor pharmaco­kinetic profile, due to short in vivo half-life. Therefore, ex­tending the in vivo half-life of peptides is clearly desirable in order for their therapeutic potential to be realized, without the need for high doses and frequent administration. Our group is developing a novel strategy that will enhance the pharmacokinetic properties of thera­peutic peptides, which could decrease dosing frequency and im­prove patient convenience and compliance

The Cis-Δ2,3-Double Bond of Phoslactomycins is Generated by a Post-PKS Tailoring Enzyme

The antifungal phoslactomycins (PLM A-F), produced by Streptomyes sp HK803, are structurally unusual in that three of their four double bonds are in the cis form (Δ12,13, Δ14,15, Δ2,3). The PLM polyketide synthase (PKS) has the predicted dehydratase catalytic domain in modules 1,2 and 5 required for establishing two of these cis double bonds (Δ12,13Δ14,15), as well as the only trans Δ6,7double bond. By contrast, the formation of the cis Δ2,3 in the unsaturated lactone moiety of PLMs has presented an enigma because the predicted dehydratase domain in module 7 is absent. Herein, we have demonstrated that the plmT2 gene product, with no homology to PKS dehydratase domains, is required for efficient formation of the cis Δ2,3 alkene. A series of new PLM products in which the C3 hydroxyl group is retained, are made in plmT2 deletion mutants. In all of these cases, however, the hydroxyl group is esterified with malonic acid. These malonylated PLM products are converted to the corresponding cis Δ2,3 PLM products and acetic acid by a facile base-catalyzed decarboxylative elimination reaction. Complete or partial restoration of natural PLM production in a plmT2deletion mutant can be accomplished by plasmid based expression of plmT2 or fos ORF4 (a homologous gene from the fostriecin biosynthetic gene cluster), respectively. The data indicate that dehydratase-independent pathways also function in establishment of unsaturated 6-membered lactone moieties in other PKS pathways, and provide the first biosynthetic insights into the possible routes by which unusual malonylated polyketide products are generated