Directed Biosynthesis of Iso-aclacinomycins with Improved Anticancer Activity.

A four-enzyme catalyzed hydroxy regioisomerization of anthracycline was integrated into the biosynthetic pathway of aclacinomycin A (ALM-A), to generate a series of iso-ALMs via directed combinatorial biosynthesis combined with precursor-directed mutasynthesis. Most of the newly acquired iso-ALMs exhibit obviously (1-5-fold) improved antitumor activity. Therefore, we not only developed iso-ALMs with potential as clinical drugs but also demonstrated the utility of this tailoring tool for modification of anthracycline antibiotics in drug discovery and development.

Characterization of rhodosaminyl transfer by the AknS/AknT glycosylation complex and its use in reconstituting the biosynthetic pathway of aclacinomycin A.

The tetracyclic core of anthracycline natural products with antitumor activity such as aclacinomycin A are tailored during biosynthesis by regioselective glycosylation. We report the first synthesis of TDP-L-rhodosamine and demonstrate that the glycosyltransferase AknS transfers L-rhodosamine to the aglycone to initiate construction of the side-chain trisaccharide. The partner protein AknT accelerates AknS turnover rate for L-rhodosamine transfer by 200-fold. AknT does not affect the Km but rather affects the kcat. Using these data, we propose that AknT causes a conformational change in AknS that stabilizes the transition state and ultimately enhances transfer. When the subsequent glycosyltransferase AknK and its substrate TDP-L-fucose are also added to the aglycone, the disaccharide and low levels of a fully reconstituted trisaccharide form of aclacinomycin are observed.

Aclacinomycin oxidoreductase (AknOx) from the biosynthetic pathway of the antibiotic aclacinomycin is an unusual flavoenzyme with a dual active site.

Aclacinomycin (Acl) oxidoreductase (AknOx) catalyzes the last two steps in the biosynthesis of polyketide antibiotics of the Acl group, the oxidation of the terminal sugar moiety rhodinose to l-aculose. We present the crystal structure of AknOx with bound FAD and the product AclY, refined to 1.65-A resolution. The overall fold of AknOx identifies the enzyme as a member of the p-cresol methylhydroxylase superfamily. The cofactor is bicovalently attached to His-70 and Cys-130 as 8alpha-Ndelta1-histidyl, 6-S-cysteinyl FAD. The polyketide ligand is bound in a deep cleft in the substrate-binding domain, with the tetracyclic ring system close to the enzyme surface and the three-sugar chain extending into the protein interior. The terminal sugar residue packs against the isoalloxazine ring of FAD and positions the carbon atoms that are oxidized close to the N5 atom of FAD. The structure and site-directed mutagenesis data presented here are consistent with a mechanism where the two different reactions of AknOx are catalyzed in the same active site but by different active site residues. Tyr-450 is responsible for proton removal from the C-4 hydroxyl group in the first reaction, the oxidation of rhodinose to cinerulose A. Tyr-378 acts as a catalytic base involved in proton abstraction from C3 of cinerulose A in the second reaction, for formation L-aculose. Replacement of this residue, however, does not impair the conversion of rhodinose to cinerulose A.

Structure determination by multiwavelength anomalous diffraction of aclacinomycin oxidoreductase: indications of multidomain pseudomerohedral twinning.

The crystal structure of aclacinomycin oxidoreductase (AknOx), a tailoring enzyme involved in the biosynthesis of the polyketide antibiotic aclacinomycin, was determined to 1.65 A resolution by multiwavelength anomalous diffraction using data from selenomethionine-substituted crystals. The crystals belong to space group P2(1), with unit-cell parameters a = 68.2, b = 264.5, c = 68.2 A, beta = 119 degrees . Analysis of the intensity statistics clearly showed the presence of pseudomerohedral twinning. The data set could also be indexed and scaled with an R(sym) of 0.072 in the orthorhombic space group C222(1) (unit-cell parameters a = 69.7, b = 117.5, c = 264.4 A), indicating the possibility of pseudomerohedral twinning along the diagonal between the monoclinic a and c directions. Refinement using this twin operator resulted in an R(free) of 24.2%. A monoclinic lattice with a = c and beta close to 120 degrees can emulate a hexagonal metric, with the possibility of a threefold twin operator along the b axis and three twin domains. Refinement assuming three-domain twinning gave a final R(free) of 26.5%. The structure of AknOx can be thus refined with comparable R(free) values using either of the twin operators separately, suggesting the possibility that crystals of AknOx contain six twin domains generated by the twofold and threefold twin operators perpendicular to each other. Both twin operators coincide with noncrystallographic symmetry axes that may promote twinning.

Crystal structure of the polyketide cyclase AknH with bound substrate and product analogue: implications for catalytic mechanism and product stereoselectivity.

AknH is a small polyketide cyclase that catalyses the closure of the fourth carbon ring in aclacinomycin biosynthesis in Streptomyces galilaeus, converting aklanonic acid methyl ester to aklaviketone. The crystal structure analysis of this enzyme, in complex with substrate and product analogue, showed that it is closely related in fold and mechanism to the polyketide cyclase SnoaL that catalyses the corresponding reaction in the biosynthesis of nogalamycin. Similarity is also apparent at a functional level as AknH can convert nogalonic acid methyl ester, the natural substrate of SnoaL, to auraviketone in vitro and in constructs in vivo. Despite the conserved structural and mechanistic features between these enzymes, the reaction products of AknH and SnoaL are stereochemically distinct. Supplied with the same substrate, AknH yields a C9-R product, like most members of this family of polyketide cyclases, whereas the product of SnoaL has the opposite C9-S stereochemistry. A comparison of high-resolution crystal structures of the two enzymes combined with in vitro mutagenesis studies revealed two critical amino acid substitutions in the active sites, which contribute to product stereoselectivity in AknH. Replacement of residues Tyr15 and Asn51 of AknH, located in the vicinity of the main catalytic residue Asp121, by their SnoaL counter-parts phenylalanine and leucine, respectively, results in a complete loss of product stereoselectivity.

Engineered biosynthesis of aklanonic acid analogues.

Aklanonic acid, an anthraquinone natural product, is a common advanced intermediate in the biosynthesis of several antitumor polyketide antibiotics, including doxorubicin and aclacinomycin A. Intensive semisynthetic and biosynthetic efforts have been directed toward developing improved analogues of these clinically important compounds. The primer unit of such polyfunctional aromatic polyketides is an attractive site for introducing novel chemical functionality, and attempts have been made to modify the primer unit by precursor-directed biosynthesis or protein engineering of the polyketide synthase (PKS). We have previously demonstrated the feasibility of engineering bimodular aromatic PKSs capable of synthesizing unnatural hexaketides and octaketides. In this report, we extend this ability by preparing analogues of aklanonic acid, a decaketide, and its methyl ester. For example, by recombining the R1128 initiation module with the dodecaketide-specific pradimicin PKS, the isobutyryl-primed analogue of aklanonic acid (YT296b, 10) and its methyl ester (YT299b, 12) were prepared. In contrast, elongation modules from dodecaketide-specific spore pigment PKSs were unable to interact with the R1128 initiation module. Thus, in addition to revealing a practical route to new anthracycline antibiotics, we also observed a fundamental incompatibility between antibiotic and spore pigment biosynthesis in the actinomycetes bacteria.

AknK is an L-2-deoxyfucosyltransferase in the biosynthesis of the anthracycline aclacinomycin A.

The antitumor drug aclacinomycin A is a representative member of the anthracycline subgroup that contains a C(7)-O-trisaccharide chain composed of L-2-deoxysugars. The sugar portion of the molecule, which greatly affects its biological activity, is assembled by dedicated glycosyltransferases; however, these enzymes have not been well-studied. Here we report the heterologous expression and purification of one of these enzymes, AknK, as well as the preparation of dTDP-L-2-deoxysugar donors, dTDP-L-2-deoxyfucose and dTDP-L-daunosamine, and the monoglycosyl aglycone, rhodosaminyl aklavinone. Our experiments show that AknK catalyzes the addition of the second sugar to the chain, using dTDP-L-2-deoxyfucose and rhodosaminyl aklavinone, to create the L-2-deoxyfucosyl-L-rhodosaminyl aklavinone. AknK also accepts an alternate dTDP-L-sugar, dTDP-L-daunosamine, and other monoglycosylated anthracyclines, including daunomycin, adriamycin, and idarubicin, to build alternate disaccharides on variant anthracycline backbones. Remarkably, AknK also catalyzes a tandem addition of a second L-2-deoxyfucosyl moiety, albeit with reduced activity, to the natural disaccharide chain to produce L-deoxyfucosyl-L-deoxyfucosyl-L-rhodosaminyl aklavinone, a variant of the natural aclacinomycin A. These results demonstrate that AknK may be a useful enzyme for the chemoenzymatic synthesis of anthracycline variants.

Engineering anthracycline biosynthesis toward angucyclines.

The biosynthesis pathways of two anthracyclines, nogalamycin and aclacinomycin, were directed toward angucyclines by using an angucycline-specific cyclase, pgaF, isolated from a silent antibiotic biosynthesis gene cluster. Addition of pgaF to a gene cassette that harbored the early biosynthesis genes of nogalamycin resulted in the production of two known angucyclinone metabolites, rabelomycin and its precursor, UWM6. Substrate flexibility of pgaF was demonstrated by replacement of the nogalamycin minimal polyketide synthase genes in the gene cassette with the equivalent aclacinomycin genes together with aknE2 and aknF, which specify the unusual propionate starter unit in aclacinomycin biosynthesis. This modification led to the production of a novel angucyclinone, MM2002, in which the expected ethyl side chain was incorporated into the fourth ring.

Cloning and characterization of Streptomyces galilaeus aclacinomycins polyketide synthase (PKS) cluster.

We have cloned and sequenced polyketide synthase (PKS) genes from the aclacinomycin producer Streptomyces galilaeus ATCC 31,615. The sequenced 13.5-kb region contained 13 complete genes. Their organization as well as their protein sequences showed high similarity to those of other type II PKS genes. The continuous region included the genes for the minimal PKS, consisting of ketosynthase I (aknB), ketosynthase II (aknC), and acyl carrier protein (aknD). These were followed by the daunomycin dpsC and dpsD homologues (aknE2 and F, respectively), which are rare in type II PKS clusters. They are associated with the unusual starter unit, propionate, used in the biosynthesis of aklavinone, a common precursor of aclacinomycin and daunomycin. Accordingly, when aclacinomycins minimal PKS genes were substituted for those of nogalamycin in the plasmid carrying genes for auramycinone biosynthesis, aklavinone was produced in the heterologous hosts. In addition to the minimal PKS, the cloned region included the PKS genes for polyketide ketoreductase (aknA), aromatase (aknE1) and oxygenase (aknX), as well as genes putatively encoding an aklanonic acid methyl transferase (aknG) and an aklanonic acid methyl ester cyclase (aknH) for post-polyketide steps were found. Moreover, the region carried genes for an activator (aknI), a glycosyl transferase (aknK) and an epimerase (aknL) taking part in deoxysugar biosynthesis.

A gene cluster from Streptomyces galilaeus involved in glycosylation of aclarubicin.

We have cloned and characterized a gene cluster for anthracycline biosynthesis from Streptomyces galilaeus. This cluster, 15-kb long, includes eight genes involved in the deoxyhexose biosynthesis pathway, a gene for a glycosyltransferase and one for an activator, as well as two genes involved in aglycone biosynthesis. Gene disruption targeted to the activator gene blocked production of aclacinomycins in S. galilaeus. Plasmid pSgs4, containing genes for a glycosyltransferase (aknS), an aminomethylase (aknX), a glucose-1-phosphate thymidylyltransferase (akn Y) and two genes for unidentified glycosylation functions (aknT and aknV), restored the production of aclacinomycins in the S. galilaeus mutants H063, which accumulates aklavinone, and H054, which produces aklavinone with rhodinose and deoxyfucose residues. Furthermore, pSgs4 directed the production of L-rhamnosyl-epsilon-rhodomycinone and L-daunosaminyl-epsilon-rhodomycinone in S. peucetius strains that produce epsilon-rhodomycinone endogenously. Subcloning of the gene cluster was carried out in order to further define the genes that are responsible for complementation and hybrid anthracycline generation.

Folding of the polyketide chain is not dictated by minimal polyketide synthase in the biosynthesis of mithramycin and anthracycline.

BACKGROUND: Mithramycin, nogalamycin and aclacinomycins are aromatic polyketide antibiotics that exhibit antitumour activity. The precursors of these antibiotics are formed via a polyketide biosynthetic pathway in which acetate (for mithramycinone and nogalamycinone) or propionate (for aklavinone) is used as a starter unit and nine acetates are used as extender units. The assembly of building blocks is catalyzed by the minimal polyketide synthase (PKS). Further steps include regiospecific reductions (if any) and cyclization. In the biosynthesis of mithramycin, however, ketoreduction is omitted and the regiospecificity of the first cyclization differs from that of anthracycline antibiotics (e.g. nogalamycin and aclacinomycins). These significant differences provide a convenient means to analyze the determinants for the regiospecificity of the first cyclization step. RESULTS: In order to analyze a possible role of the minimal PKS in the regiospecificity of the first cyclization in polyketide biosynthesis, we expressed the mtm locus, which includes mithramycin minimal PKS genes, in Streptomyces galilaeus, which normally makes aclacinomycins, and the sno locus, which includes nogalamycin minimal PKS genes, in Streptomyces argillaceus, which normally makes mithramycin. The host strains are defective in the minimal PKS, but they express other antibiotic biosynthesis genes. Expression of the sno minimal PKS in the S. argillaceus polyketide-deficient strain generated mithramycin production. Auramycins, instead of aclacinomycins, accumulated in the recombinant S. galilaeus strains, suggesting that the mithramycin minimal PKS is responsible for the choice of starter unit. We also describe structural analysis of the compounds accumulated by a ketoreductase-deficient S. galilaeus mutant; spectroscopic studies on the major polyketide compound that accumulated revealed a first ring closure which is not typical of anthracyclines, suggesting an important role for the ketoreductase in the regiospecificity of the first cyclization. CONCLUSIONS: These experiments clearly support the involvement of ketoreductase and a cyclase in the regiospecific cyclization of the biosynthetic pathway for aromatic polyketides.

Minimal Streptomyces sp. strain C5 daunorubicin polyketide biosynthesis genes required for aklanonic acid biosynthesis.

The structure of the Streptomyces sp. strain C5 daunorubicin type II polyketide synthase (PKS) gene region is different from that of other known type II PKS gene clusters. Directly downstream of the genes encoding ketoacylsynthase alpha and beta (KS alpha, KS beta) are two genes (dpsC, dpsD) encoding proteins of unproven function, both absent from other type II PKS gene clusters. Also in contrast to other type II PKS clusters, the gene encoding the acyl carrier protein (ACP), dpsG, is located about 6.8 kbp upstream of the genes encoding the daunorubicin KS alpha and KS beta. In this work, we demonstrate that the minimal genes required to produce aklanonic acid in heterologous hosts are dpsG (ACP), dauI (regulatory activator), dpsA (KS alpha), dpsB (KS beta), dpsF (aromatase), dpsE (polyketide reductase), and dauG (putative deoxyaklanonic acid oxygenase). The two unusual open reading frames, dpsC (KASIII homolog lacking a known active site) and dpsD (acyltransferase homolog), are not required to synthesize aklanonic acid. Additionally, replacement of dpsD or dpsCD in Streptomyces sp. strain C5 with a neomycin resistance gene (aphI) results in mutant strains that still produced anthracyclines.

Aclacinomycin X, a novel anthracycline antibiotic produced by Streptomyces galilaeus ATCC 31133.

A new anthracycline antibiotic, designated as aclacinomycin X, was isolated from the culture broth of Streptomyces galilaeus ATCC 31133, and was identified as 7-(O-rhodosaminyl-deoxyfucosyl-rednosyl)- aklavinone. Its in vitro cytotoxicity was tested against several human tumor cell lines.

Expression of Streptomyces peucetius genes for doxorubicin resistance and aklavinone 11-hydroxylase in Streptomyces galilaeus ATCC 31133 and production of a hybrid aclacinomycin.

The aklavinone 11-hydroxylase gene and two doxorubicin resistance genes cloned from Streptomyces peucetius subsp. caesius ATCC 27952 were introduced into doxorubicin-sensitive Streptomyces galilaeus ATCC 31133, an aclacinomycin producer. The doxorubicin resistance genes drrA and drrB endowed S. galilaeus with high-level resistance to doxorubicin, indicating that the resistance mechanism for doxorubicin might be different from that for aclacinomycin A. Transformation of S. galilaeus ATCC 31133 with plasmid pMC213 containing the aklavinone 11-hydroxylase gene (dnrF) resulted in the production of many red pigments. A new metabolite was purified, and the position of the newly introduced hydroxyl group was determined. This result indicated that the aklavinone 11-hydroxylase gene was stably expressed in S. galilaeus ATCC 31133 and that it gave rise to a hybrid aclacinomycin A which showed highly specific in vitro cytotoxicity against leukemia and melanoma cell lines.

Isolation and characterization of aclacinomycin A-non-producing Streptomyces galilaeus (ATCC 31615) mutants.

Twelve mutants of Streptomyces galilaeus (ATCC 31615) blocked in the production of aclacinomycin A, an anthracycline antibiotic with significant antitumour activity, accumulated intermediates of the biosynthesis of aclacinomycins and several anthracyclines with variant sugar moieties. Three of these aklavinone glycosides have not been described before. Mutant strains H028, H061 and H036 were blocked before the formation of aklavinone, a common intermediate for most anthracyclines. Strain H039 accumulated aklavinone and H026, H035, H038 and H054 had mutations that changed glycosylation of aklavinone. Characterization of the mutants and their products is described.

Microbial glycosidation of some anthracycline antibiotics by an antibiotic-negative mutant of aclarubicin producer.

Microbial conversion of anthracyclinone monosaccharides using aclarubicin-negative mutant of Streptomyces galilaeus was found to produce anthracyclinone disaccharides which had either rhodinose or 2-deoxyfucose as an additional sugar. By this conversion we obtained twelve new anthracyclines from seven anthracyclines which had rhodosamine, N-monomethyldaunosamine or daunosamine at C-7 as a glycosidic sugar. All products had a reduced cytotoxic activity in comparison with those of parent compounds. However, some of them showed a therapeutically improved antitumor effects against L1210 leukemia in vivo.

[Studies on the antitumor antibiotic aclacinomycin A].

During the course of screening for new antitumor antibiotics, a new anthracycline antibiotic--aclacinomycin A was separated from the broth and mycelium of Streptomyces AC-57. The strain AC-57 was isolated from the soil collected in the Shanghai suburbs. According to its culture and physiological characteristics the producer was identified as Str. galilaeus AC-57. The broth and mycelium were extracted and treated with solvents as usual way. The aclacinomycin A was separated by silica-gel column chromatography eluted with chrolo-form-methanol. Aclacinomycin A, its aglycone and sugar components were identified by comparison of their physico-chemical and spectral data (MS, UV, IR, 1H-NMR, and 13C-NMR) with authentic compound, purified from the market sample.

Compartmentation of enzymes interconverting aclacinomycins in Streptomyces species AM 33352.

The enzymatic interconversion of the aclacinomycins A (I), Y (II), and B (III) by Streptomyces spec. AM 33352/S 182 producing these aklavinone glycosides was investigated. The enzymes converting I to II and III, as well as vice versa, are located within different compartments separated by the cytoplasmic membrane. Aclacinomycin A (I) is biotransformed to II and III by the cell-free mycelium extract while the entire mycelium carries out the same type of conversion towards the opposite direction. Changes of enzyme activity are correlated to alterations in the ratio of aklavinone glycosides throughout the fermentation. A hypothesis is developed concerning the role of compartmentized oxidoreductase(s) in the passive flux of I from inside the cells to outside.