Enediyne Antitumor Antibiotic C-1027 Research Articles

Structural Insights into the Free-Standing Condensation Enzyme SgcC5 Catalyzing Ester-Bond Formation in the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027

C-1027 is a chromoprotein enediyne antitumor antibiotic, consisting of the CagA apoprotein and the C-1027 chromophore. The C-1027 chromophore features a nine-membered enediyne core appended with three peripheral moieties, including an ( S)-3-chloro-5-hydroxy-β-tyrosine. In a convergent biosynthesis of the C-1027 chromophore, the ( S)-3-chloro-5-hydroxy-β-tyrosine moiety is appended to the enediyne core by the free-standing condensation enzyme SgcC5. Unlike canonical condensation domains from the modular nonribosomal peptide synthetases that catalyze amide-bond formation, SgcC5 catalyzes ester-bond formation, as demonstrated in vitro, between SgcC2-tethered ( S)-3-chloro-5-hydroxy-β-tyrosine and ( R)-1-phenyl-1,2-ethanediol, a mimic of the enediyne core as an acceptor substrate. Here, we report that (i) genes encoding SgcC5 homologues are widespread among both experimentally confirmed and bioinformatically predicted enediyne biosynthetic gene clusters, forming a new clade of condensation enzymes, (ii) SgcC5 shares a similar overall structure with the canonical condensation domains but forms a homodimer in solution, the active site of which is located in a cavity rather than a tunnel typically seen in condensation domains, and (iii) the catalytic histidine of SgcC5 activates the 2-hydroxyl group, while a hydrogen-bond network in SgcC5 prefers the R-enantiomer of the acceptor substrate, accounting for the regio- and stereospecific ester-bond formation between SgcC2-tethered ( S)-3-chloro-5-hydroxy-β-tyrosine and ( R)-1-phenyl-1,2-ethanediol upon acid-base catalysis. These findings expand the catalytic repertoire and reveal new insights into the structure and mechanism of condensation enzymes.

Discovery of Alternative Producers of the Enediyne Antitumor Antibiotic C-1027 with High Titers.

The potent cytotoxicity and unique mode of action make the enediyne antitumor antibiotic C-1027 an exquisite drug candidate for anticancer chemotherapy. However, clinical development of C-1027 has been hampered by its low titer from the original producer Streptomyces globisporus C-1027. Here we report three new C-1027 alternative producers, Streptomyces sp. CB00657, CB02329, and CB03608, from The Scripps Research Institute actinomycetes strain collection. Together with the previously disclosed Streptomyces sp. CB02366 strain, four C-1027 alternative producers with C-1027 titers of up to 11-fold higher than the original producer have been discovered. The five C-1027 producers, isolated from distant geographic locations, are distinct Streptomyces strains based on morphology and taxonomy. Pulsed-field gel electrophoresis and Southern analysis of the five C-1027 producers reveal that their C-1027 biosynthetic gene clusters (BGCs) are all located on giant plasmids of varying sizes. The high nucleotide sequence similarity among the five C-1027 BGCs implies that they most likely have evolved from a common ancestor.

Crystal Structures of SgcE6 and SgcC, the Two-Component Monooxygenase That Catalyzes Hydroxylation of a Carrier Protein-Tethered Substrate during the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027 in Streptomyces globisporus.

C-1027 is a chromoprotein enediyne antitumor antibiotic produced by Streptomyces globisporus. In the last step of biosynthesis of the (S)-3-chloro-5-hydroxy-β-tyrosine moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose a two-component monooxygenase that hydroxylates the C-5 position of (S)-3-chloro-β-tyrosine. This two-component monooxygenase is remarkable for two reasons. (i) SgcE6 specifically reacts with FAD and NADH, and (ii) SgcC is active with only the peptidyl carrier protein (PCP)-tethered substrate. To address the molecular details of substrate specificity, we determined the crystal structures of SgcE6 and SgcC at 1.66 and 2.63 Å resolution, respectively. SgcE6 shares a similar β-barrel fold with the class I HpaC-like flavin reductases. A flexible loop near the active site of SgcE6 plays a role in FAD binding, likely by providing sufficient space to accommodate the AMP moiety of FAD, when compared to that of FMN-utilizing homologues. SgcC shows structural similarity to a few other known FADH2-dependent monooxygenases and sheds light on some biochemically but not structurally characterized homologues. The crystal structures reported here provide insights into substrate specificity, and comparison with homologues provides a catalytic mechanism of the two-component, FADH2-dependent monooxygenase (SgcE6 and SgcC) that catalyzes the hydroxylation of a PCP-tethered substrate.

Crystal structure of SgcJ, an NTF2-like superfamily protein involved in biosynthesis of the nine-membered enediyne antitumor antibiotic C-1027.

Comparative analysis of the enediyne biosynthetic gene clusters revealed sets of conserved genes serving as outstanding candidates for the enediyne core. Here we report the crystal structures of SgcJ and its homologue NCS-Orf16, together with gene inactivation and site-directed mutagenesis studies, to gain insight into enediyne core biosynthesis. Gene inactivation in vivo establishes that SgcJ is required for C-1027 production in Streptomyces globisporus. SgcJ and NCS-Orf16 share a common structure with the nuclear transport factor 2-like superfamily of proteins, featuring a putative substrate binding or catalytic active site. Site-directed mutagenesis of the conserved residues lining this site allowed us to propose that SgcJ and its homologues may play a catalytic role in transforming the linear polyene intermediate, along with other enediyne polyketide synthase associated enzymes, into an enzyme-sequestered enediyne core intermediate. These findings will help formulate hypotheses and design experiments to ascertain the function of SgcJ and its homologues in 9-membered enediyne core biosynthesis.

Characterization of SfmD as a Heme Peroxidase That Catalyzes the Regioselective Hydroxylation of 3-Methyltyrosine to 3-Hydroxy-5-methyltyrosine in Saframycin A Biosynthesis

Saframycin A (SFM-A) is a potent antitumor antibiotic that belongs to the tetrahydroisoquinoline family. Biosynthetic studies have revealed that its unique pentacyclic core structure is derived from alanine, glycine, and non-proteinogenic amino acid 3-hydroxy-5-methyl-O-methyltyrosine (3-OH-5-Me-OMe-Tyr). SfmD, a hypothetical protein in the biosynthetic pathway of SFM-A, was hypothesized to be responsible for the generation of the 3-hydroxy group of 3-OH-5-Me-OMe-Tyr based on previously heterologous expression results. We now report the in vitro characterization of SfmD as a novel heme-containing peroxidase that catalyzes the hydroxylation of 3-methyltyrosine to 3-hydroxy-5-methyltyrosine using hydrogen peroxide as the oxidant. In addition, we elucidated the biosynthetic pathway of 3-OH-5-Me-OMe-Tyr by kinetic studies of SfmD in combination with biochemical assays of SfmM2, a methyltransferase within the same pathway. Furthermore, SacD, a counterpart of SfmD involved in safracin B biosynthesis, was also characterized as a heme-containing peroxidase, suggesting that SfmD-like heme-containing peroxidases may be commonly involved in the biosynthesis of SFM-A and its analogs. Finally, we found that the conserved motif HXXXC is crucial for heme binding using comparative UV-Vis and Magnetic Circular Dichroism (MCD) spectra studies of SfmD wild-type and mutants. Together, these findings expand the category of heme-containing peroxidases and set the stage for further mechanistic studies. In addition, this study has critical implications for delineating the biosynthetic pathway of other related tetrahydroisoquinoline family members.

Probing the active site of MIO-dependent aminomutases, key catalysts in the biosynthesis of beta-amino acids incorporated in secondary metabolites.

The tyrosine aminomutase SgTAM produces (S)-ss-tyrosine from L-tyrosine in the biosynthesis of the enediyne antitumor antibiotic C-1027. This conversion is promoted by the methylideneimidazole-5-one (MIO) prosthetic group. MIO was first identified in the homologous family of ammonia lyases, which deaminate aromatic amino acids to form alpha,ss-unsaturated carboxylates. Studies of substrate specificity have been described for lyases but there have been limited reports in altering the substrate specificity of aminomutases. Furthermore, it remains unclear as to what structural properties are responsible for catalyzing the presumed readdition of the amino group into the alpha,ss-unsaturated intermediates to form ss-amino acids. Attempts to elucidate specificity and mechanistic determinants of SgTAM have also proved to be difficult as it is recalcitrant to perturbations to the active site via mutagenesis. An X-ray cocrystal structure of the SgTAM mutant of the catalytic base with L-tyrosine verified important substrate binding residues as well as the enzymatic base. Further mutagenesis revealed that removal of these crucial interactions renders the enzyme inactive. Proposed structural determinants for mutase activity probed via mutagenesis, time-point assays and X-ray crystallography revealed a complicated role for these residues in maintaining key quaternary structure properties that aid in catalysis.

Manipulation of pathway regulation in Streptomyces globisporus for overproduction of the enediyne antitumor antibiotic C-1027

Manipulation of pathway regulation is an efficient strategy to increase specific secondary metabolite production. Here we successfully improved production of both the enediyne antitumor antibiotic C-1027 and a heptaebe, an early metabolite of the C-1027 pathway, by manipulating the three regulatory genes, sgcR1, sgcR2, and sgcR3, within the C-1027 biosynthetic gene cluster. SgcR3 has previously been established as an activator, and we now propose that SgcR1 and SgcR2 are also positive regulators based on their up-regulation effects on titer and/or timing of heptaene and C-1027 production in Streptomyces globisporus. Specifically, overexpression of sgcR1 significantly improved production of the heptaene (about 5-fold) and C-1027 (2- to 3-fold) compared to the wild-type strain. However, the titers of heptaene and C-1027 were not increased by overexpressing all three activators together, underscoring the complexity of C-1027 biosynthetic pathway regulation. The possibility of exploiting the heptaene as a readily identifiable and unique indicator for rapidly detecting enediyne production was also assessed.

Characterization of SgcE6, the flavin reductase component supporting FAD-dependent halogenation and hydroxylation in the biosynthesis of the enediyne antitumor antibiotic C-1027

The C-1027 enediyne antitumor antibiotic from Streptomyces globisporus possesses an (S)-3-chloro-5-hydroxy-beta-tyrosine moiety, the chloro- and hydroxy-substituents of which are installed by a flavin-dependent halogenase SgcC3 and monooxygenase SgcC, respectively. Interestingly, a single flavin reductase, SgcE6, can provide reduced flavin to both enzymes. Bioinformatics analysis reveals that, similar to other flavin reductases involved in natural product biosynthesis, SgcE6 belongs to the HpaC-like subfamily of the Class I flavin reductases. The present study describes the steady-state kinetic characterization of SgcE6 as a strictly NADH- and FAD-specific enzyme.

Biosynthesis of the enediyne antitumor antibiotic C-1027 involves a new branching point in chorismate metabolism

C-1027 is an enediyne antitumor antibiotic composed of four distinct moieties: an enediyne core, a deoxy aminosugar, a beta-amino acid, and a benzoxazolinate moiety. We now show that the benzoxazolinate moiety is derived from chorismate by the sequential action of two enzymes-SgcD, a 2-amino-2-deoxyisochorismate (ADIC) synthase and SgcG, an iron-sulfur, FMN-dependent ADIC dehydrogenase-to generate 3-enolpyruvoylanthranilate (OPA), a new intermediate in chorismate metabolism. The functional elucidation and catalytic properties of each enzyme are described, including spectroscopic characterization of the products and the development of a fluorescence-based assay for kinetic analysis. SgcD joins isochorismate (IC) synthase and 4-amino-4-deoxychorismate (ADC) synthase as anthranilate synthase component I (ASI) homologues that are devoid of pyruvate lyase activity inherent in ASI; yet, in contrast to IC and ADC synthase, SgcD has retained the ability to aminate chorismate identically to that observed for ASI. The net conversion of chorismate to OPA by the tandem action of SgcD and SgcG unambiguously establishes a new branching point in chorismate metabolism.

The Structure of l-Tyrosine 2,3-Aminomutase from the C-1027 Enediyne Antitumor Antibiotic Biosynthetic Pathway,

The SgcC4 l-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)-beta-tyrosine in the biosynthetic pathway of the enediyne antitumor antibiotic C-1027. SgTAM is homologous to the histidine ammonia lyase family of enzymes whose activity is dependent on the methylideneimidazole-5-one (MIO) cofactor. Unlike the lyase enzymes, SgTAM catalyzes additional chemical transformations resulting in an overall stereospecific 1,2-amino shift in the substrate l-tyrosine to generate (S)-beta-tyrosine. Previously, we provided kinetic, spectroscopic, and mutagenesis data supporting the presence of MIO in the active site of SgTAM [Christenson, S. D.; Wu, W.; Spies, A.; Shen, B.; and Toney, M. D. (2003) Biochemistry 42, 12708-12718]. Here we report the first X-ray crystal structure of an MIO-containing aminomutase, SgTAM, and confirm the structural homology of SgTAM to ammonia lyases. Comparison of the structure of SgTAM to the l-tyrosine ammonia lyase from Rhodobacter sphaeroides provides insight into the structural basis for aminomutase activity. The results show that SgTAM has a closed active site well suited to retain ammonia and minimize the formation of lyase elimination products. The amino acid determinants for substrate recognition and catalysis can be predicted from the structure, setting the framework for detailed mechanistic investigations.

Substrate Specificity of the Adenylation Enzyme SgcC1 Involved in the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027

C-1027 is an enediyne antitumor antibiotic composed of a chromophore with four distinct chemical moieties, including an (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety that is derived from l-alpha-tyrosine. SgcC4, a novel aminomutase requiring no added co-factor that catalyzes the formation of the first intermediate (S)-beta-tyrosine and subsequently SgcC1 homologous to adenylation domains of nonribosomal peptide synthetases, was identified as specific for the SgcC4 product and did not recognize any alpha-amino acids. To definitively establish the substrate for SgcC1, a full kinetic characterization of the enzyme was performed using amino acid-dependent ATP-[(32)P]PP(i) exchange assay to monitor amino acid activation and electrospray ionization-Fourier transform mass spectroscopy to follow the loading of the activated beta-amino acid substrate to the peptidyl carrier protein SgcC2. The data establish (S)-beta-tyrosine as the preferred substrate, although SgcC1 shows promiscuous activity toward aromatic beta-amino acids such as beta-phenylalanine, 3-chloro-beta-tyrosine, and 3-hydroxy-beta-tyrosine, but all were <50-fold efficient. A putative active site mutant P571A adjacent to the invariant aspartic acid residue of all alpha-amino acid-specific adenylation domains known to date was prepared as a preliminary attempt to probe the substrate specificity of SgcC1; however the mutation resulted in a loss of activity with all substrates except (S)-beta-tyrosine, which was 142-fold less efficient relative to the wild-type enzyme. In total, SgcC1 is now confirmed to catalyze the second step in the biosynthesis of the (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety of C-1027, presenting downstream enzymes with an (S)-beta-tyrosyl-S-SgcC2 thioester substrate, and represents the first beta-amino acid-specific adenylation enzyme characterized biochemically.

Biosynthesis of the β-Amino Acid Moiety of the Enediyne Antitumor Antibiotic C-1027 Featuring β-Amino Acyl-S-carrier Protein Intermediates

The enediyne antitumor antibiotic C-1027 chromoprotein is produced by Streptomyces globisporus. The biosynthesis of the (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety (boxed) of the C-1027 chromophore (1) from l-tyrosine (3) and its incorporation into 1 are catalyzed by six enzymes: SgcC, SgcC1, SgcC2, SgcC3, SgcC4, ShcC5. In vivo and in vitro characterization of these enzymes delineated this pathway, unveiling a novel strategy for beta-amino acid modification featuring beta-amino acyl-S-carrier protein intermediates. These findings shed new light into beta-amino acid biosynthesis and present a new opportunity to engineer the C-1027 biosynthetic machinery for the production of novel analogues as exemplified by 20-deschloro-C-1027 (4), 20-deschro-22-deshydroxy-C-1027 (5), and 22-deshydroxy-C-1027 (6).

Biochemical characterization of the SgcA1 alpha-D-glucopyranosyl-1-phosphate thymidylyltransferase from the enediyne antitumor antibiotic C-1027 biosynthetic pathway and overexpression of sgcA1 in Streptomyces globisporus to improve C-1027 production.

Sequence analysis of the biosynthetic gene cluster for the enediyne antitumor antibiotic C-1027 from Streptomyces globisporus has previously suggested that the sgcA1 gene encodes a alpha-d-glucopyranosyl-1-phosphate thymidylyltransferase (Glc-1-P-TT) catalyzing the first step in the biosynthesis of the 4-deoxy-4-(dimethylamino)-5,5-dimethyl-d-ribopyranose moiety by activating alpha-d-glucopyranosyl-1-phosphate (Glc-1-P) into deoxythymidine diphosphate-alpha-d-glucose (dTDP-Glc). Here we report the overexpression of sgcA1 in E. coli, purification of the overproduced SgcA1 to homogenetity, biochemical and kinetic characterization of the purified SgcA1 as a Glc-1-P-TT, and yield improvement for C-1027 production by overexpression of sgcA1 and its flanking gene in S. globisporus. These findings provide biochemical evidence supporting the genetics-based hypothesis for C-1027 biosynthesis, set the stage for further investigation of the deoxysugar biosynthetic pathway, and demonstrate the utility of sugar biosynthesis genes in natural product yield improvement via combinatorial biosynthesis methods. In contrast to the homotetrameric quaternary structure known for Glc-1-P-TT enzymes from primary metabolic pathways, Glc-1-P-TT enzymes such as SgcA1 from secondary metabolic pathways are monomeric in solution. Sequence differences between the two subclasses of Glc-1-P-TT enzymes were noted. The monomeric structural feature of the latter enzymes could be exploited in engineering Glc-1-P-TT enzymes with broad substrate specificity for structural diversity via the glycorandomization strategy.

Kinetic Analysis of the 4-Methylideneimidazole-5-one-Containing Tyrosine Aminomutase in Enediyne Antitumor Antibiotic C-1027 Biosynthesis

The enediyne antitumor antibiotic C-1027 contains an unusual (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety, which requires an aminomutase for its biosynthesis. Previously, we established that SgcC4 is an aminomutase that catalyzes the conversion of L-tyrosine to (S)-beta-tyrosine and employs 4-methylideneimidazole-5-one (MIO) at its active site [Christenson, S. D., Liu, W., Toney, M. D., and Shen, B. (2003) J. Am. Chem. Soc. 125, 6062-6063]. Here, we present a thorough analysis of the properties of SgcC4. L-Tyrosine is the best substrate among those tested and most likely serves as the in vivo precursor for the (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety. The presence of MIO in the active site is supported by several lines of evidence. (1) Addition of ATP or divalent metal ions has no effect on its aminomutase activity. (2) SgcC4 has optimal activity at pH approximately 8.8, similar to the pH optima of MIO-dependent ammonia lyases. (3) SgcC4 is strongly inhibited by sodium borohydride and potassium cyanide, but preincubation with L-tyrosine or 4-hydroxycinnamate largely prevents this inhibition. (4) The difference spectrum between SgcC4 and its S153A mutant shows a positive peak at approximately 310 nm, indicative of MIO. (5) The S153A mutation lowers k(cat)/K(M) 640-fold. The SgcC4-catalyzed conversion of L-tyrosine to (S)-beta-tyrosine proceeds via 4-hydroxycinnamate as an intermediate. The latter also acts as a competitive inhibitor with respect to L-tyrosine and serves as an alternative substrate for the production of beta-tyrosine in the presence of an amino source. A full time course for the SgcC4-catalyzed interconversion between L-tyrosine, beta-tyrosine, and 4-hydroxycinnamate was measured and analyzed to provide estimates for the rate constants in a minimal mechanism. SgcC4 also exhibits a beta-tyrosine racemase activity, but alpha-tyrosine racemase activity was not detected.

Mechanism of Formation of Novel Covalent Drug&middot;DNA Interstrand Cross-Links and Monoadducts by Enediyne Antitumor Antibiotics

The potent enediyne antitumor antibiotic C1027 has been previously reported to induce novel DNA interstrand cross-links and drug monoadducts under anaerobic conditions [Xu et al. (1997) J. Am. Chem. Soc. 119, 1133-1134]. In the present study, we explored the mechanism of formation of these anaerobic DNA lesions. We found that, similar to the aerobic reaction, the diradical species of the activated drug initiates anaerobic DNA damage by abstracting hydrogen atoms from the C4', C1', and C5' positions of the A1, A2, and A3 nucleotides, respectively, in the most preferred 5'GTTA1T/5'ATA2A3C binding sequence. It is proposed that the newly generated deoxyribosyl radicals, which cannot undergo oxidation, likely add back onto the nearby unsaturated ring system of the postactivated enediyne core, inducing the formation of interstrand cross-links, connecting either A1 to A2 or A1 to A3, or drug monoadducts mainly on A2 or A3. Comparative studies with other enediynes, such as neocarzinostatin and calicheamicin gamma1I under similar reaction conditions indicate that the anaerobic reaction process is a kinetically competitive one, depending on the proximity of the drug unsaturated ring system or dioxygen to the sugar radicals and their quenching by other hydrogen sources such as solvent or thiols. It was found that C1027 mainly generates interstrand cross-links, whereas most of the anaerobic lesions produced by neocarzinostatin are drug monoadducts. Calicheamicin gamma1I was found to be less efficient in producing both lesions. The anaerobic DNA lesions induced by enediyne antitumor antibiotics may have important implications for their potent cytotoxicity in the central regions of large tumors, where relative anaerobic conditions prevail.

Induction of Apoptosis by Enediyne Antitumor Antibiotic C1027 in HL-60 Human Promyelocytic Leukemia Cells

C1027, a new macromolecular antitumor antibiotic with an enediyne chromophore, displays extremely potent cytotoxicity against cancer cells. HL-60 human promyelocytic leukemia cells treated with C1027 (0.1-10 nM) for 2 hours resulted in morphological changes, including Hoechst 33342-stained condensed nuclei, condensation of nuclear chromatin, and nuclear fragmentation. Agarose gel electrophoresis of C1027-treated HL-60 cells showed a typical ladder-like pattern of DNA fragments. In addition, the apoptotic DNA peak of propidium iodide-stained nuclei was revealed by flow cytometry. Treatment of HL-60 cells with C1027 (5 nM) induced apoptosis in up to 79% of the cells. The results suggest that C1027 may exert antitumor activity by triggering apoptosis.