Biomedical subjects

David H Sherman

Publications and source records attributed to David H Sherman.

At least 37 records · Page 2Linked to original sources

PubMed2005

Biochemical investigation of pikromycin biosynthesis employing native penta- and hexaketide chain elongation intermediates.

The unique ability of the pikromycin (Pik) polyketide synthase to generate 12- and 14-membered ring macrolactones presents an opportunity to explore the fundamental processes underlying polyketide synthesis, specifically the mechanistic details of chain extension, keto group processing, acyl chain release, and macrocyclization. We have synthesized the natural pentaketide and hexaketide chain elongation intermediates as N-acetyl cysteamine (NAC) thioesters and have used them as substrates for in vitro conversions with engineered PikAIII+TE and in combination with native PikAIII (module 5) and PikAIV (module 6) multifunctional proteins. This investigation demonstrates directly the remarkable ability of these monomodules to catalyze one or two chain extension reactions, keto group processing steps, acyl-ACP release, and cyclization to generate 10-deoxymethynolide and narbonolide. The results reveal the enormous preference of Pik monomodules for their natural polyketide substrates and provide an important comparative analysis with previous studies using unnatural diketide NAC thioester substrates.

Amino Acid Sequence↗

PubMed2005

Exploiting the reaction flexibility of a type III polyketide synthase through in vitro pathway manipulation.

A synthetic metabolic pathway has been constructed in vitro consisting of the type III polyketide synthase from Streptomyces coelicolor and peroxidases from soybean and Caldariomyces fumago (chloroperoxidase). This has resulted in the synthesis of the pentaketide flaviolin and its dimeric derivative, and a wide range of pyrones and their coupled derivatives with flaviolin, as well as their halogenated derivatives. The addition of acyl-CoA oxidase to the pathway prior to the polyketide synthase resulted in unsaturated pyrone side chains, further broadening the product spectrum that can be achieved. The approach developed in this work, therefore, provides a new model to exploit biocatalysis in the synthesis of complex natural product derivatives.

Acyl Coenzyme A↗

PubMed2005

Utilizing the power of microbial genetics to bridge the gap between the promise and the application of marine natural products.

Marine organisms are a rich source of secondary metabolites. They have yielded thousands of compounds with a broad range of biomedical applications. Thus far, samples required for preclinical and clinical studies have been obtained by collection from the wild, by mariculture, and by total chemical synthesis. However, for a number of complex marine metabolites, none of these options is feasible for either economic or environmental reasons. In order to proceed with the development of many of these promising therapeutic compounds, a reliable and renewable source must be found. Over the last twenty years, the study of microbial secondary metabolites has greatly advanced our understanding of how nature utilizes simple starting materials to yield complex small molecules. Much of this work has focused on polyketides and nonribosomal peptides, two classes of molecules that are prevalent in marine micro- and macroorganisms. The lessons learned from the study of terrestrial metabolite biosynthesis are now being applied to the marine world. As techniques for cloning and heterologous expression of biosynthetic pathways continue to improve, they may provide our greatest hope for bridging the gap between the promise and application of many marine natural products.

Animals↗

PubMed2005

The Lego-ization of polyketide biosynthesis.

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Catalysis↗

PubMed2005

Molecular analysis of the rebeccamycin L-amino acid oxidase from Lechevalieria aerocolonigenes ATCC 39243.

Rebeccamycin, a member of the tryptophan-derived indolocarbazole family, is produced by Lechevalieria aerocolonigenes ATCC 39243. The biosynthetic pathway that specifies biosynthesis of this important metabolite is comprised of 11 genes spanning 18 kb of DNA. A presumed early enzyme involved in elaboration of the rebeccamycin aglycone is encoded by rebO, located at the left-hand region of the reb gene cluster. The deduced protein product, RebO (51.9 kDa), is an L-amino acid oxidase (L-AAO) that has 27% identity to an L-AAO from Scomber japonicus (animal, mackerel) and is a member of the family of FAD-dependent oxidase enzymes. In order to study the biochemical properties of this key enzyme, the rebO gene was overexpressed and purified from Escherichia coli. Biochemical characterization showed that RebO is dimeric, with a molecular mass of approximately 101 kDa. Further analysis revealed that the enzyme contains a noncovalently bound FAD cofactor and is reoxidized at the expense of molecular oxygen by producing one molecule of hydrogen peroxide. Based on kinetic studies, RebO shows significant preference for 7-chloro-L-tryptophan, suggesting its likely role as the natural early pathway substrate. Furthermore, the native RebO enzyme has evident, albeit limited, flexibility as shown by bioconversion studies with unnatural substrates. This work provides the first analysis of a structural enzyme involved in construction of this important class of indolocarbazole natural products.

Actinomycetales↗

PubMed2004

Bacterial P450-catalyzed polyketide hydroxylation on a microfluidic platform.

The incorporation of a multicomponent, cofactor-dependant P450 into a microfluidic biochip is demonstrated. The PikC hydroxylase Streptomyces venezuelae was incorporated into a PDMS-based microfluidic channel. The enzyme was immobilized to Ni-NTA agarose beads via in situ attachment following the addition of the beads to the microchannel. The enzyme loading was approximately 6 microg per mg of beads resulting in a microchannel loading of 10.7 mg/mL. This high enzyme loading enabled the rapid hydroxylation of the macrolide YC-17 to methymycin and neomethymycin in about equal amounts with a conversion of >90% at a flow rate of 70 nL/min. This high reactivity allowed rapid hydroxylation reactions to be performed with short residence times, which is critical for complex enzymes with limited inherent stability.

Bioreactors↗

PubMed2004

An efficient method for creation and functional analysis of libraries of hybrid type I polyketide synthases.

Bacterial type I polyketide synthases (PKSs) generate a structurally diverse group of natural products with a wide range of biological activities. Hybrid type I PKSs in which domains of one multifunctional polypeptide are replaced with components from heterologous systems have generated significant interest over the past decade. Almost invariably only one or several specific hybrids are made at a time and tested for functionality. This approach is slow, dependent upon a fortuitous choice of specific fusions points, and often leads to inactive or minimally active hybrid systems. We describe herein a method for generating and screening a library of hybrid pikAI complementation plasmids (encoding the loading domain and the first two extension domains of pikromycin PKS) able to restore pikromycin in a BB138 Streptomyces venezuelae pikAI-deletion mutant. In the first step the plasmid sequence encoding the loading domain AT(0)-ACP(0) was replaced by a counter selectable marker, sacB. DNA family shuffling was then used to generate a diverse library of chimeric AT(0)-ACP(0) fragments, which were used to replace sacB by lambda-Red-mediated in vivo recombination in an Escherichia coli host. This method resulted in the rapid and efficient generation of a large number of hybrid pikAI complementation plasmids, which were used to transform S.venezuelae BB138. A bioassay of over 4000 of these transformants successfully revealed three different PikAI hybrids which were able to lead to pikromycin production. The study suggests that most of the hybrids are not detectably functional, and underscores the need to generate and screen large and diverse libraries in which different fusion points are tried. The methodologies applied in this study address this need and can be used for directed evolution of any component of the PikPKS, and potentially other type I PKS systems.

Acyltransferases↗

PubMed2004

bryA: an unusual modular polyketide synthase gene from the uncultivated bacterial symbiont of the marine bryozoan Bugula neritina.

"Candidatus Endobugula sertula," the uncultivated bacterial symbiont of Bugula neritina, is the proposed source of the bryostatin family of anticancer compounds. We cloned a large modular polyketide synthase (PKS) gene complex from "Candidatus Endobugula sertula" and characterized one gene, bryA, which we propose is responsible for the initial steps of bryostatin biosynthesis. Typical PKS domains are present. However, acyltransferase domains are lacking in bryA, and beta-ketoacyl synthase domains of bryA cluster with those of PKSs with discrete, rather than integral, acyltransferases. We propose a model for biosynthesis of the bryostatin D-lactate starter unit by the bryA loading module, utilizing atypical domains homologous to FkbH, KR, and DH. The bryA gene product is proposed to synthesize a portion of the pharmacologically active part of bryostatin and may be useful in semisynthesis of clinically useful bryostatin analogs.

Amino Acid Sequence↗

PubMed2004

Biosynthetic pathway and gene cluster analysis of curacin A, an antitubulin natural product from the tropical marine cyanobacterium Lyngbya majuscula.

Curacin A (1) is a potent cancer cell toxin obtained from strains of the tropical marine cyanobacterium Lyngbya majuscula found in Curaçao. Its structure is unique in that it contains the sequential positioning of a thiazoline and cyclopropyl ring, and it exerts its potent cell toxicity through interaction with the colchicine drug binding site on microtubules. A series of stable isotope-labeled precursors were fed to cultures of curacin A-producing strains and, following NMR analysis, allowed determination of the metabolic origin of all atoms in the natural product (one cysteine, 10 acetate units, two S-adenosyl methionine-derived methyl groups) as well as several unique mechanistic insights. Moreover, these incorporation experiments facilitated an effective gene cloning strategy that allowed identification and sequencing of the approximately 64 kb putative curacin A gene cluster. The metabolic system is comprised of a nonribosomal peptide synthetase (NRPS) and multiple polyketide synthases (PKSs) and shows a very high level of collinearity between genes in the cluster and the predicted biochemical steps required for curacin biosynthesis. Unique features of the cluster include (1) all but one of the PKSs are monomodular multifunctional proteins, (2) a unique gene cassette that contains an HMG-CoA synthase likely responsible for formation of the cyclopropyl ring, and (3) a terminating motif that is predicted to function in both product release and terminal dehydrative decarboxylation.

Amino Acid Sequence↗

PubMed2004

Comparison of the 1.85 A structure of CYP154A1 from Streptomyces coelicolor A3(2) with the closely related CYP154C1 and CYPs from antibiotic biosynthetic pathways.

The genus Streptomyces produces two-thirds of microbially derived antibiotics. Polyketides form the largest and most diverse group of these natural products. Antibiotic diversity of polyketides is generated during their biosynthesis by several means, including postpolyketide modification performed by oxidoreductases, a broad group of enzymes including cytochrome P450 monooxygenases (CYPs). CYPs catalyze site-specific oxidation of macrolide antibiotic precursors significantly affecting antibiotic activity. Efficient manipulation of Streptomyces CYPs in generating new antibiotics will require identification and/or engineering of monooxygenases with activities toward a diverse array of chemical substrates. To begin to link structure to function of CYPs involved in secondary metabolic pathways of industrially important species, we determined the X-ray structure of Streptomyces coelicolor A3(2) CYP154A1 at 1.85 A and analyzed it in the context of the closely related CYP154C1 and more distant CYPs from polyketide synthase (EryF) and nonribosomal peptide synthetase (OxyB) biosynthetic pathways. In contrast to CYP154C1, CYP154A1 reveals an active site inaccessible from the molecular surface, and an absence of catalytic activities observed for CYP154C1. Systematic variations in the amino acid patterns and length of the surface HI loop correlate with degree of rotation of the F and G helices relative to the active site in CYP154A1-related CYPs, presumably regulating the degree of active site accessibility and its dimensions. Heme in CYP154A1 is in a 180 degrees flipped orientation compared with most other structurally determined CYPs.

Amino Acid Sequence↗

PubMed2004

Isolation and characterization of novel marine-derived actinomycete taxa rich in bioactive metabolites.

A unique selective enrichment procedure has resulted in the isolation and identification of two new genera of marine-derived actinobacteria. Approximately 90% of the microorganisms cultured by using the presented method were from the prospective new genera, a result indicative of its high selectivity. In this study, 102 actinomycetes were isolated from subtidal marine sediments collected from the Bismarck Sea and the Solomon Sea off the coast of Papua New Guinea. A combination of physiological parameters, chemotaxonomic characteristics, distinguishing 16S rRNA gene sequences, and phylogenetic analysis based on 16S rRNA genes provided strong evidence for the two new genera (represented by strains of the PNG1 clade and strain UMM518) within the family Micromonosporaceae. Biological activity testing of fermentation products from the new marine-derived actinomycetes revealed that several had activities against multidrug-resistant gram-positive pathogens, malignant cells, and vaccinia virus replication.

Anti-Bacterial Agents↗

PubMed2003

Merging the potential of microbial genetics with biological and chemical diversity: an even brighter future for marine natural product drug discovery.

Marine invertebrates and a growing number of marine bacteria are the sources of novel, bioactive secondary metabolites. Structurally, many of these compounds appear to be biosynthesized by polyketide synthases (PKS) and/or nonribosomal peptide synthetases (NRPS) that have also been found in terrestrial microbes. This review highlights scientific advances from 1999-2003 in the emerging field of molecular genetics of polyketide and nonribosomal peptide natural products isolated from marine organisms. The implications of this research towards the development of marine secondary metabolites as a sustainable source of new drugs are discussed.

Biodiversity↗

PubMed2003

Substrate recognition and channeling of monomodules from the pikromycin polyketide synthase.

Bacillus subtilis↗

PubMed2003

Iterative chain elongation by a pikromycin monomodular polyketide synthase.

The unique ability of the pikromycin polyketide synthase (Pik PKS) to generate 12- and 14-membered ring macrolactones presents an opportunity to explore the fundamental processes of polyketide synthesis, specifically, the mechanistic details of the chain extension process. We have overexpressed and purified PikAIII and PikAIV and demonstrated the ability of these proteins to generate triketide lactone products using (14)C-methylmalonyl-CoA as the sole substrate. Monomodular PikAIII generates TKL (1) when reacted alone, and synthesizes TKL (2) upon reaction in combination with PikAIV. Product formation remains dependent on the enzymatic decarboxylation of methylmalonyl-CoA and transfer of the acyl chain within the enzyme rather than acylation by propionyl-CoA from spontaneous decarboxylation. We propose that synthesis of TKL (1) by PikAIII involves iterative assembly of the triketide chain within a PikAIII homodimer analogous to the nonmodular type I PKS systems.

Anti-Bacterial Agents↗

PubMed2003

The 1.92-A structure of Streptomyces coelicolor A3(2) CYP154C1. A new monooxygenase that functionalizes macrolide ring systems.

Evolutionary links between cytochrome P450 monooxygenases, a superfamily of extraordinarily divergent heme-thiolate proteins catalyzing a wide array of NADPH/NADH- and O(2)-dependent reactions, are becoming better understood because of availability of an increasing number of fully sequenced genomes. Among other reactions, P450s catalyze the site-specific oxidation of the precursors to macrolide antibiotics in the genus Streptomyces introducing regiochemical diversity into the macrolide ring system, thereby significantly increasing antibiotic activity. Developing effective uses for Streptomyces enzymes in biosynthetic processes and bioremediation requires identification and engineering of additional monooxygenases with activities toward a diverse array of small molecules. To elucidate the molecular basis for substrate specificity of oxidative enzymes toward macrolide antibiotics, the x-ray structure of CYP154C1 from Streptomyces coelicolor A3(2) was determined (Protein Data Bank code ). Relocation of certain common P450 secondary structure elements, along with a novel structural feature involving an additional beta-strand transforming the five-stranded beta-sheet into a six-stranded variant, creates an open cleft-shaped substrate-binding site between the two P450 domains. High sequence similarity to macrolide monooxygenases from other microbial species translates into catalytic activity of CYP154C1 toward both 12- and 14-membered ring macrolactones in vitro.

Anti-Bacterial Agents↗

PubMed2002

Biochemical evidence for an editing role of thioesterase II in the biosynthesis of the polyketide pikromycin.

The pikromycin biosynthetic gene cluster contains the pikAV gene encoding a type II thioesterase (TEII). TEII is not responsible for polyketide termination and cyclization, and its biosynthetic role has been unclear. During polyketide biosynthesis, extender units such as methylmalonyl acyl carrier protein (ACP) may prematurely decarboxylate to generate the corresponding acyl-ACP, which cannot be used as a substrate in the condensing reaction by the corresponding ketosynthase domain, rendering the polyketide synthase module inactive. It has been proposed that TEII may serve as an "editing" enzyme and reactivate these modules by removing acyl moieties attached to ACP domains. Using a purified recombinant TEII we have tested this hypothesis by using in vitro enzyme assays and a range of acyl-ACP, malonyl-ACP, and methylmalonyl-ACP substrates derived from either PikAIII or the loading didomain of DEBS1 (6-deoxyerythronolide B synthase; AT(L)-ACP(L)). The pikromycin TEII exhibited high K(m) values (>100 microm) with all substrates and no apparent ACP specificity, catalyzing cleavage of methylmalonyl-ACP from both AT(L)-ACP(L) (k(cat)/K(m) 3.3 +/- 1.1 m(-1) s(-1)) and PikAIII (k(cat)/K(m) 2.9 +/- 0.9 m(-1) s(-1)). The TEII exhibited some acyl-group specificity, catalyzing hydrolysis of propionyl (k(cat)/K(m) 15.8 +/- 1.8 m(-1) s(-1)) and butyryl (k(cat)/K(m) 17.5 +/- 2.1 m(-1) s(-1)) derivatives of AT(L)-ACP(L) faster than acetyl (k(cat)/K(m) 4.9 +/- 0.7 m(-1) s(-1)), malonyl (k(cat)/K(m) 3.9 +/- 0.5 m(-1) s(-1)), or methylmalonyl derivatives. PikAIV containing a TEI domain catalyzed cleavage of propionyl derivative of AT(L)-ACP(L) at a dramatically lower rate than TEII. These results provide the first unequivocal in vitro evidence that TEII can hydrolyze acyl-ACP thioesters and a model for the action of TEII in which the enzyme remains primarily dissociated from the polyketide synthase, preferentially removing aberrant acyl-ACP species with long half-lives. The lack of rigorous substrate specificity for TEII may explain the surprising observation that high level expression of the protein in Streptomyces venezuelae leads to significant (>50%) titer decreases.

Anti-Bacterial Agents↗

PubMed2002

An unexpected interaction between the modular polyketide synthases, erythromycin DEBS1 and pikromycin PikAIV, leads to efficient triketide lactone synthesis.

An unusual feature of the 6-module pikromycin polyketide synthase (PikPKS, PikAI-PikAIV) of S. venezuelae is the ability to generate both 12- and 14-membered ring macrolides. The PikAIV component containing the last extension module and a thioesterase domain is responsible for generating both of these products. In the case of the 12-membered ring macrolide, an acyl-enzyme intermediate on PikAIII is able to efficiently "skip" the last extension step and is cyclized by the TE domain of PikAIV, presumably as a result of a PikAIII-PikAIV interaction. Herein we report that plasmid-based expression (pBK3) of DEBS1, which comprises the loading domain and the first two modules of the Saccharopolyspora erythrea 6-deoxyerythronolide B synthase, in S. venezuelae leads to efficient 15 +/- 3 mg/L production of triketide lactone products (TKLs). Comparable levels of TKLs were observed with a plasmid (pBK1) which expressed DEBS1 fused to a TE domain (DEBS1-TE). These results are in stark contrast to previous in vivo and in vitro analyses, where only DEBS1-TE efficiently produces TKLs. Levels of TKLs decreased dramatically with expression of DEBS1 in both pikAIV and pikAIII-pikAIV deletion hosts (0.5 mg/L), but not DEBS1-TE, and could be partially restored by addition of a PikAIV complementation plasmid. These data suggest that PikAIV is able to efficiently catalyze formation of 6-membered lactone ring products from acyl-bound intermediates on DEBS1 in a manner analogous to that observed for 12-membered macrolide products from PikAIII. Significant sequence similarity and length of the C-terminal linker region of PikAIII and DEBS1 suggest that this region may be responsible for the interaction with PikAIV. A replacement of this linker region of DEBS1 with the corresponding region of PikAI led to a 95% decrease in TKL levels in S. venezuelae, consistent with this hypothesis.

Amino Acid Sequence↗

PubMed2002

The barbamide biosynthetic gene cluster: a novel marine cyanobacterial system of mixed polyketide synthase (PKS)-non-ribosomal peptide synthetase (NRPS) origin involving an unusual trichloroleucyl starter unit.

Barbamide was extracted from the marine cyanobacterium Lyngbya majuscula strain 19L as a chlorinated lipopeptide for its potent molluscicidal activity. Precursor incorporation studies indicated that it is derived from acetate, L-phenylalanine, L-leucine and L-cysteine. The gene cluster responsible for biosynthesis of barbamide (bar) was cloned and characterized in this study. DNA sequence analysis of cosmid pLM49 revealed a cluster of 12 open reading frames (barA-barK) extending 26 kb including the expected polyketide synthase and non-ribosomal peptide synthetase modules and tailoring genes. The genetic architecture and domain organization of the bar cluster supports the assignment based on the apparent co-linearity of the systems. The activity assay of adenylation domains of barD (A(D)), barE (A(E)) and barG (A(G2) for module 2) in an amino acid-dependent ATP-pyrophosphate exchange experiment supports the conclusion that barbamide is synthesized from acetate, L-phenylalanine, L-cysteine and L-leucine with trichloroleucine as a direct precursor by a mixed polyketide synthase/non-ribosomal polypeptide synthetase. Assembly of barbamide includes unique biochemical mechanisms for chlorination, one-carbon truncation during chain elongation, E-double bond formation and thiazole ring formation.

Adenosine Triphosphate↗