Alessandra S. Eustáquio

Biosynthesis of the salinosporamide A polyketide synthase substrate chloroethylmalonyl-coenzyme A from S-adenosyl-l-methionine

Polyketides are among the major classes of bioactive natural products used to treat microbial infections, cancer, and other diseases. Here we describe a pathway to chloroethylmalonyl-CoA as a polyketide synthase building block in the biosynthesis of salinosporamide A, a marine microbial metabolite whose chlorine atom is crucial for potent proteasome inhibition and anticancer activity. S-adenosyl-l-methionine (SAM) is converted to 5′-chloro-5′-deoxyadenosine (5′-ClDA) in a reaction catalyzed by a SAM-dependent chlorinase as previously reported. By using a combination of gene deletions, biochemical analyses, and chemical complementation experiments with putative intermediates, we now provide evidence that 5′-ClDA is converted to chloroethylmalonyl-CoA in a 7-step route via the penultimate intermediate 4-chlorocrotonyl-CoA. Because halogenation often increases the bioactivity of drugs, the availability of a halogenated polyketide building block may be useful in molecular engineering approaches toward polyketide scaffolds.

Engineering Fluorometabolite Production: Fluorinase Expression in Salinispora tropica Yields Fluorosalinosporamide†

Organofluorine compounds play an important role in medicinal chemistry, where they are responsible for up to 15% of the pharmaceutical products on the market. While natural products are valuable sources of new chemical entities, natural fluorinated molecules are extremely rare and the pharmaceutical industry has not benefited from a microbial source of this class of compounds. Streptomyces cattleya is an unusual bacterium in that it elaborates fluoroacetate and the amino acid 4-fluorothreonine. The discovery in 2002 of the fluorination enzyme FlA responsible for C-F bond formation in S. cattleya, and its subsequent characterization, opened up for the first time the prospect of genetically engineering fluorometabolite production from fluoride ion in host organisms. As a proof of principle, we report here the induced production of fluorosalinosporamide by replacing the chlorinase gene salL from Salinispora tropica with the fluorinase gene flA.

Advances in and applications of proteasome inhibitors.

With the recent US Food and Drug Administration approval of bortezomib (Velcade) for the treatment of relapsed multiple myeloma, the proteasome has emerged as a new therapeutic target with diverse pathology. Drug discovery programs in academia and the pharmaceutical industry have developed a range of low nanomolar synthetic and natural inhibitors of the 20S proteasome core particle that have entered human clinical trials as significant anti-cancer and anti-inflammatory leads. Moreover, proteasome inhibitors continue to serve as valuable research tools in cellular biology through the elucidation of important biological processes associated with the ubiquitin-proteasome pathway of protein degradation. This review will highlight recent advances in the development and application of proteasome inhibitors.

Significant Natural Product Biosynthetic Potential of Actinorhizal Symbionts of the Genus Frankia, as Revealed by Comparative Genomic and Proteomic Analyses

ABSTRACT Bacteria of the genus Frankia are mycelium-forming actinomycetes that are found as nitrogen-fixing facultative symbionts of actinorhizal plants. Although soil-dwelling actinomycetes are well-known producers of bioactive compounds, the genus Frankia has largely gone uninvestigated for this potential. Bioinformatic analysis of the genome sequences of Frankia strains ACN14a, CcI3, and EAN1pec revealed an unexpected number of secondary metabolic biosynthesis gene clusters. Our analysis led to the identification of at least 65 biosynthetic gene clusters, the vast majority of which appear to be unique and for which products have not been observed or characterized. More than 25 secondary metabolite structures or structure fragments were predicted, and these are expected to include cyclic peptides, siderophores, pigments, signaling molecules, and specialized lipids. Outside the hopanoid gene locus, no cluster could be convincingly demonstrated to be responsible for the few secondary metabolites previously isolated from other Frankia strains. Few clusters were shared among the three species, demonstrating species-specific biosynthetic diversity. Proteomic analysis of Frankia sp. strains CcI3 and EAN1pec showed that significant and diverse secondary metabolic activity was expressed in laboratory cultures. In addition, several prominent signals in the mass range of peptide natural products were observed in Frankia sp. CcI3 by intact-cell matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). This work supports the value of bioinformatic investigation in natural products biosynthesis using genomic information and presents a clear roadmap for natural products discovery in the Frankia genus.

Biosynthesis of Salinosporamides from α,β-Unsaturated Fatty Acids: Implications for Extending Polyketide Synthase Diversity

A new series of coenzyme A-tethered polyketide synthase extender units were discovered in relation to the biosynthesis of the salinosporamide family of anticancer agents from the marine bacterium Salinispora tropica. In vivo and in vitro experiments revealed that the crotonyl-CoA reductase/carboxylase SalG has broad substrate tolerance toward 2-alkenyl-CoAs that give rise to the salinosporamide C-2 substitution pattern.

The Discovery of Salinosporamide K from the Marine Bacterium “Salinispora pacifica” by Genome Mining Gives Insight into Pathway Evolution

The γ-lactam-β-lactone natural product salinosporamide A (1) is a potent proteasome inhibitor produced by the marine bacterium Salinispora tropica.[1,2] This chlorinated anticancer agent dominates a family of natural structural analogues that primarily differ at the C-2 substituent.[3] In the case of 1, the C-2 chloroethyl group is a key functional group that enables the molecule to irreversibly bind to the 20S proteasome.[4] All γ-lactam-β-lactone natural products, including the salinosporamides (1–4), the cinnabaramides (5), and omuralide (6), share the initial reaction with the proteasome in which the N-terminal threo-nine residue of the catalytic β-subunit attacks the β-lactone group of the inhibitor to form an ester linkage.[3] While this covalent proteasome–inhibitor complex is susceptible to water hydrolysis, a subsequent reaction of the β-lactone-derived C-3 hydroxyl group with a C-2 side-chain leaving group such as in 1 yields a tetrahydrofuran adduct that is stable to hydrolysis.[4] Due to the mechanistic importance of the salinosporamide C-2 substituent, biosynthetic studies in S. tropica explored the origins of this small compound library to reveal that the salinosporamides are atypical products of a hybrid polyketide synthase–nonribosomal peptide synthetase (PKS–NRPS).[5,6] By accommodating different PKS building blocks such as chloroethyl-, methyl-, ethyl-, and propyl-malonyl-CoA, salinosporamides A(1), D (2), B (3) and E (4), respectively, are biosynthesized.[6,7] This understanding provided the logic to engineer the unnatural derivative fluorosalinosporamide[8,9] (7) as well as the molecular basis to explore new genome sequences for the discovery of novel salinosporamide derivatives. Herein we report the genome-inspired discovery and characterization of salinosporamide K (8) from a new source, “Salinispora pacifica” strain CNT-133, that provides insight into the evolution of the salinosporamide biosynthetic pathway. From the three proposed Salinispora species, S. tropica and “S. pacifica” are more closely related to each other than each is to S. arenicola.[10]

Enzymatic Halogenation and Dehalogenation Reactions: Pervasive and Mechanistically Diverse

Naturally produced halogenated compounds are ubiquitous across all domains of life where they perform a multitude of biological functions and adopt a diversity of chemical structures. Accordingly, a diverse collection of enzyme catalysts to install and remove halogens from organic scaffolds has evolved in nature. Accounting for the different chemical properties of the four halogen atoms (fluorine, chlorine, bromine, and iodine) and the diversity and chemical reactivity of their organic substrates, enzymes performing biosynthetic and degradative halogenation chemistry utilize numerous mechanistic strategies involving oxidation, reduction, and substitution. Biosynthetic halogenation reactions range from simple aromatic substitutions to stereoselective C-H functionalizations on remote carbon centers and can initiate the formation of simple to complex ring structures. Dehalogenating enzymes, on the other hand, are best known for removing halogen atoms from man-made organohalogens, yet also function naturally, albeit rarely, in metabolic pathways. This review details the scope and mechanism of natures halogenation and dehalogenation enzymatic strategies, highlights gaps in our understanding, and posits where new advances in the field might arise in the near future.

Discovery and Assembly Line Biosynthesis of the Lymphostin Pyrroloquinoline Alkaloid Family of mTOR Inhibitors in Salinispora Bacteria

The pyrroloquinoline alkaloid family of natural products, which includes the immunosuppressant lymphostin, has long been postulated to arise from tryptophan. We now report the molecular basis of lymphostin biosynthesis in three marine Salinispora species that maintain conserved biosynthetic gene clusters harboring a hybrid nonribosomal peptide synthetase-polyketide synthase that is central to lymphostin assembly. Through a series of experiments involving gene mutations, stable isotope profiling, and natural product discovery, we report the assembly-line biosynthesis of lymphostin and nine new analogues that exhibit potent mTOR inhibitory activity.

Novobiocin biosynthesis: inactivation of the putative regulatory gene novE and heterologous expression of genes involved in aminocoumarin ring formation

The left ends of the biosynthetic gene clusters of novobiocin (nov), clorobiocin (clo) and coumermycin A1 (cou) from Streptomyces spheroides (syn. S. caeruleus) NCIMB 11891, S. roseochromogenes var. oscitans DS 12.976 and S. rishiriensis DSM 40489 were cloned and sequenced. Sequence comparison suggested that novE, cloE and couE, respectively, represent the borders of these three clusters. Inactivation of novE proved that novE does not have an essential catalytic role in novobiocin biosynthesis, but is likely to have a regulatory function. The gene products of novF and cloF show sequence similarity to prephenate dehydrogenase and may produce 4-hydroxyphenylpyruvate (4HPP) as a precursor of the substituted benzoate moiety of novobiocin and clorobiocin. Coumermycin A1 does not contain this benzoate moiety, and correspondingly the coumermycin cluster was found not to contain a functional novF homologue. The coumermycin biosynthetic gene cluster apparently evolved from an ancestral cluster similar to those of novobiocin and clorobiocin, and parts of the ancestral novF homologue have been deleted in this process. No homologue to novC was identified in the gene clusters of clorobiocin and coumermycin, questioning the postulated involvement of novC in aminocoumarin biosynthesis. Heterologous expression of novDEFGHIJK in Streptomyces lividans resulted in the formation of 2,4-dihydroxy-α-oxy-phenylacetic acid, suggesting that at least one of the proteins encoded by these genes may participate in a hydroxylation reaction.

Evaluation of Streptomyces coelicolor A3(2) as a heterologous expression host for the cyanobacterial protein kinase C activator lyngbyatoxin A

Filamentous marine cyanobacteria are extremely rich sources of bioactive natural products and often employ highly unusual biosynthetic enzymes in their assembly. However, the current lack of techniques for stable DNA transfer into these filamentous organisms, combined with the absence of heterologous expression strategies for nonribosomal cyanobacterial gene clusters, prohibit the creation of mutant strains or the heterologous production of these cyanobacterial compounds in other bacteria. In this study, we evaluated the capability of a derivative of the model actinomycete Streptomyces coelicolor A3(2) to express enzymes involved in the biosynthesis of the protein kinase C activator lyngbyatoxin A from a Hawaiian strain of Moorea producta (previously classified as Lyngbya majuscula). Despite large differences in GC content between these two bacteria and the presence of rare TTA/UUA leucine codons in lyngbyatoxin ORFs we were able to achieve expression of the cytochrome P450 monooxygenase LtxB and reverse prenyltransferase LtxC in S. coelicolor M512 and confirmed the in vitro functionality of S. coelicolor overexpressed LtxC. Attempts to express the entire lyngbyatoxin A gene cluster in S. coelicolor M512 were not successful because of transcript termination observed for the ltxA gene, which encodes a large nonribosomal peptide synthetase. However, these attempts did show a detectable level of cyanobacterial promoter recognition in Streptomyces. Successful expression of lyngbyatoxin A proteins in Streptomyces provides a new platform for biochemical investigation of natural product enzymes from Moorea strains.