Identification of a sugar flexible glycosyltransferase from Streptomyces olivaceus, the producer of the antitumor polyketide elloramycin

Comunidad Europea, Paln Nacional de Biotecnología del Ministerio de Educación y Ciencia de España, la Comisión de Educación Superior de Carolina del Sur y el Departamento de Defensa de los estados Unido

Crystal structure of the glycosyltransferase SnogD from the biosynthetic pathway of nogalamycin in Streptomyces nogalater

The glycosyltransferase SnogD from Streptomyces nogalater transfers a nogalamine moiety to the metabolic intermediate 3′,4′-demethoxynogalose-1-hydroxynogalamycinone during the final steps of biosynthesis of the aromatic polyketide nogalamycin. The crystal structure of recombinant SnogD, as an apo-enzyme and with a bound nucleotide, 2-deoxyuridine-5′-diphosphate, was determined to 2.6 Å resolution. Reductive methylation of SnogD was crucial for reproducible preparation of diffraction quality crystals due to creation of an additional intermolecular salt bridge between methylated lysine residue Lys384 and Glu374* from an adjacent molecule in the crystal lattice. SnogD is a dimer both in solution and in the crystal, and the enzyme subunit displays a fold characteristic of the GT-B family of glycosyltransferases. Binding of the nucleotide is associated with rearrangement of two active-site loops. Site-directed mutagenesis shows that two active-site histidine residues, His25 and His301, are critical for the glycosyltransferase activities of SnogD both in vivo and in vitro. The crystal structures and the functional data are consistent with a role for His301 in binding of the diphosphate group of the sugar donor substrate, and a function of His25 as a catalytic base in the glycosyl transfer reaction.VRPublishe

Structural biology of carbohydrate transfer and modification in natural product biosynthesis

Certain organisms, can during periods of limited resources, adapt their metabolism to enable biosynthesis of secondary metabolites, compounds that increase competitiveness and chances of survival. The subjects of this thesis are enzymes acting on carbohydrate substrates during secondary metabolism. The enzymatic attachment of carbohydrate moieties onto precursors of polyketide antibiotics such as anthracyclines, required for their biological activity, is performed by glycosyltransferases (GT). The anthracycline nogalamycin contains two carbohydrates: a nogalose moiety attached via an O-glycosidic bond to C7, and a nogalamine attached via an O-glycosidic bond to C1 and an unusual carbon-carbon bond between C2 and C5´´ of the sugar. Genetic and functional data presented in this thesis established the roles of SnogE as the GT performing the C7 O-glycosyl transfer of the nogalose moiety and SnogD as the O-GT attaching the nogalamine moiety onto the C1 carbon. The activity of SnogD was verified in vitro using recombinant protein, following establishment of a transglycosylation-like assay. The three-dimensional structure of the homo-dimeric SnogD was determined to 2.6 Å and consists of a GT-B fold. Mutagenesis of two active site residues, His25 and His301, evaluated in vitro and in vivo, suggested His25 to be the catalytic base, activating the acceptor substrate by proton abstraction from the C1-hydroxyl group. His301 provides a positive charge to stabilise the negative charge formed close to the diphosphate of the leaving group during glycosyl transfer. Genetic, functional and structural data together suggest the involvement of an additional or altogether different enzyme for the C-C bond formation. The bifunctional enzyme aldos-2-ulose dehydratase (AUDH) from Phanerochaete chrysosporium catalyses the dehydration and isomerisation of the secondary metabolites glucosone and 1,5-anhydro-D-fructose (AF) into the antimicrobial compounds cortalcerone and microthecin (Mic), respectively. The threedimensional structure of the dimeric AUDH was determined to 2.0 Å. The enzyme consists of a seven bladed ß-propeller, two cupin folds and a lectin-like domain, in a novel combination. Two structural metal ions, Mg2+ and Zn2+, are bound in loop regions. Two additional zinc ions are present at the base of two putative active sites, located in the ß-propeller and the second cupin fold. The specific removal of these zinc ions eliminated catalytic activity, proving the metal dependency of the overall reaction. The structure of AUDH in complex with the reaction intermediate ascopyrone M bound at both putative active sites, and a complex of zinc-depleted enzyme with AF bound in the cupin fold have been determined by X-ray crystallography to 2.6 and 2.8 Å resolution, respectively. These observations support the presence of two distinct active sites located 60 Å apart, partly connected by an intra-dimeric channel. The dehydration reaction most likely follows an elimination reaction with the zinc ion acting as a Lewis acid to polarise the C2 keto group of AF. Abstraction of the C3 proton by the suitably located residue His155 would generate an enol intermediate, which is stabilised by the zinc ion. Return of the proton to the C4 hydroxyl group would generate a favourable leaving group

Heterologous expression of the naphthocyclinone hydroxylase gene from Streptomyces arenae for production of novel hybrid polyketides

Streptomyces arenae produces at least four different isochromanequinone antibiotics, the naphthocyclinones, of which the β- and γ-form are active against Gram-positive bacteria. The naphthocyclinone biosynthesis gene cluster was isolated from Streptomyces arenae DSM 40737 and by sequence analysis the minimal polyketide synthase genes and several genes encoding tailoring enzymes were identified. Southern blot analysis of the naphthocyclinone gene cluster indicated that a 3.5 kb BamHI fragment located approximately 9 kb downstream of the minimal PKS genes hybridizes to the schC hydroxylase DNA probe isolated from S. halstedii. Two complete and one incomplete open reading frames were identified on this fragment. Sequence analysis revealed strong homology to the genes of the actVA region of S. coelicolor, to several (suggested) hydroxylases and a putative FMN-dependent monooxygenase. The proposed hydroxylase, encoded by ncnH, could hydroxylate aloesaponarin II, a molecule that is produced by the actinorhodin minimal polyketide synthase in combination with the actinorhodin ketoreductase, aromatase and cyclase. Furthermore, this enzyme is capable of accepting additional polyketide core structures that contain a 5-hydroxy-1,4-naphthoquinone moiety as substrates which makes it an interesting tailoring enzyme for the modification of polyketide structure

Protein expression analysis of a high-demeclocycline producing strain of Streptomyces aureofaciens and the roles of CtcH and CtcJ in demeclocycline biosynthesis

Additional file 1: Online Resource 1. Primers used in this study

Exploring the Capacity of Bacteria for Natural Product Biosynthesis

This dissertation is focused on exploring the potential of bacteria for the biosynthesis of natural products with the purposes of generating novel natural product derivatives and of improving the titer of pharmaceutically important natural products. A wide variety of compounds from various sources have been historically used in the treatment and prevention of diseases. Natural products as a major source of new drugs are extensively explored due to their huge structural diversity and promising biological activities such as antimicrobial, anticancer, antifungal, antiviral and antioxidant properties. For instance, penicillin as an early-discovered antimicrobial agent has saved millions of lives, indicating the historical importance of natural products. However, the alarming rise in the prevalence of drug resistance is a serious threat to public health and it has coincided with the decreasing supply of new antibiotics. Bacteria with a tremendous undiscovered potential have still been one of the richest sources of bioactive compounds to tackle the growing threat of antibiotic-resistant pathogens. Nevertheless, the production level of those important compounds is often quite low, and often undetectable using current analytical techniques. To expand the chemical repertoire of nature and to increase the titer of the natural products, researchers have developed various strategies, such as heterologous expression, co-cultivation of different bacteria, optimization of fermentation conditions, discovery of new species, engineering of biosynthetic enzymes, and manipulating regulatory elements. Thus, in my dissertation research, I have exploited a few of these strategies. First, I heterologously expressed some of the biosynthetic genes from the sch biosynthetic gene cluster, resulted in the production of a novel glycosylated angucycline. I was also able to generate another new glycosylated derivative of angucycline through gene disruption of tailoring enzymes. In this research, I isolated two novel angucycline derivatives and gained new insights into the glycosylation steps in the biosynthesis of Sch47554 and Sch47555. Next, I engineered the regulatory elements in Streptomyces sp. SCC-2136 through the overexpression and targeted gene disruption approaches for enhanced production of pharmaceutically important angucyclines. The highest titer of Sch47554 was achieved in Streptomyces sp. SCC-2136/ΔschA4 (27.94 mg/L), which is significantly higher than the wild type. This work thus provides an initial understanding of functional roles of regulatory elements in the biosynthesis of Sch47554 and Sch47555 and several engineered strains with enhanced production of Sch47554. Last, I isolated a carotenoid-producing endophytic bacterium from the leaves of the yew tree and optimized the fermentation conditions for an improved yield of zeaxanthin diglucoside up to 206 ± 6 mg/L. With the introduction of an additional copy of the Pscrt gene cluster through an expression plasmid, the engineered strain Pseudomonas sp. 102515/pOKF192 produced zeaxanthin diglucoside at 380 ± 12 mg/L, which is 85% higher than the parent strain. This strain holds a great potential for the production of pharmaceutically important antioxidant agent, zeaxanthin diglucoside

Elucidation of the glycosylation steps during biosynthesis of antitumor macrolides PM100117 and PM100118 and engineering for novel derivatives

This work was supported by projects INNPACTO IPT-2011-0752-900000 and BIO2015-64161-R (to J.A.S) of the Spanish Ministry of Economy and Competitiveness (MINECO

The sulfur-containing antibiotic BE-7585A: A study of its synthesis

Antibiotics are often differentiated and divided using two classes, grouped by function and structure. The molecular basis and mechanism of action of many antibiotics have been studied and identified through recent advancements in molecular biology. The main structural feature of BE-7585A is an angucycline ring. Antibiotics with this unique ring structure are known to have diverse biological functions such as antitumor, enzyme inhibitory, and blood platelet aggregation inhibitory activity [1]. Although the biological effects of BE-7585A have not been studied in detail, similar synthetic pathways have begun to be elucidated, such as urdamycin and aquayamycin by the Rohr Group [2]. However, unlike urdamycin, BE-7585A has a unique feature. It is one of few naturally occurring compounds containing a C-2 thiosugar. The incorporation of this sulfur moiety in this compound, or any other C-2 thiosugar compounds, has never been studied. Two main experiments were carried out in this work to study and elucidate part of the biosynthetic pathway of this C-2 thiosugar formation. First, a pull-down assay was performed to find the sulfur donor critical to the pathway. Second, kinetic studies of the 2-thio-trehalose-6-phosphate synthase were done to determine the km and vmax values. Although the results of the first experiment were found to be inconclusive, the kinetic parameters of the reaction catalyzed by the 2-thio-trehalose-6-phosphate synthase were determined.Biochemistr

Biosynthesis of landomycin E deoxysugar part in Streptomyces globisporus 1912: sequencing and analysis of lndZ1 and lndZ3 genes

DNA fragment of landomycin E biosynthesis gene cluster 1.5 kb in size has been completely sequenced and two open reading frames were identified. Gene lndZ1 resembles NDP-hexose-3,5-epimerase and lndZ3 is similar to NDP-hexose-4-ketoreductases. LndZ1 and LndZ3 proteins are suggested to accomplish two last catalytic steps towards deoxysugar L-rhodinose present in landomycin E carbohydrate moietyThis work was supported by grant BG-117b from Ministry of Education and Science of Ukraine (to V.F). We are gratefull to the staff of DNA sequencing facility at the Dept. of Biochemistry of Cambridge University (John Lester, Tania Mironenko, Nataliya Scott) for help in sequencing

Biosynthetic studies of thiosugar-containing natural products, BE-7585A and Lincomycin A

Sulfur is an essential element found ubiquitously in living systems. However, there exist only a few sulfur-containing sugars in nature and their biosyntheses have not been well understood. On the other hand, a wide variety of sugar derivatives commonly found in natural products are often vital components for the efficacy and specificity of their parent molecules. Elucidation of such unusual sugar biosyntheses is important both for understanding their intriguing chemical mechanisms and creating unnatural compounds by altering their biosynthetic machineries, which could potentially exhibit enhanced or novel biological activities. This dissertation describes biosynthetic studies of two thiosugar-containing natural products, BE-7585A and lincomycin A, produced by Amycolatopsis orientalis and Streptomyces lincolnensis, respectively. While the former possess a C-2-thiosugar-containing disaccharide moiety, the latter contains a C-1-thio substituent on a characteristic eight-carbon backbone sugar. The focus of this research is to characterize the biological pathways and mechanisms responsible for the sulfur incorporation and the unique sugar scaffolds. BE-7585A, an angucycline-type natural product, contains the rare C-2-thiosugar moiety. PCR-based screening of a cosmid library constructed from the genomic DNA of A. orientalis led to the identification of the BE-7585A biosynthetic gene cluster. A gene, bexX, was found to be a candidate for a thiosugar synthase with moderate sequence similarity to a thiazole synthase. The gene, bexX, and a glycosyltransferase homologue, bexG2, were heterologously expressed in Escherichia coli. A variety of biochemical experiments provided a wealth of evidence supporting the proposed biosynthetic pathway for the C-2-thiodisaccharide moiety. Finally, whole genome sequencing and a genome mining approach led to the identification of a sulfur carrier protein to accomplish the in vitro enzymatic synthesis of the C-2-thiosugar for the first time. Lincomycin A is a lincosamide antimicrobial natural product with a C-1 methylthio substituent. Although the lincomycin A biosynthetic gene cluster has been reported, biochemical verification of the biosynthetic pathway has remained elusive. In this dissertation, the complete methlthiolincosamide biosynthetic pathway including the potential C-1 sulfur incorporation mechanism was proposed. Furthermore, two early intermediates of the pathway were characterized for the first time by demonstrating the LmbR (transaldolase) and LmbN (isomerase) reactions in vitro.Chemistr