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 nine-membered enediyne core biosynthesis

Genetic and enzymatic characterization of the actinomycin biosynthesis in Streptomyces

Die Actinomycine, eine Gruppe cytostatisch wirksamer Chromopeptide, werden von verschiedenen Streptomyces Stämmen gebildet. Ihre Struktur ist geprägt durch eine Dimethyl-Phenoxazinon-Dicarbonsäure, welche in Amidbindungen mit zwei Pentapeptidlactonringen verknüpft ist. Die Bildung des Phenoxazinon-Chromophors findet im letzten Schritt der Actinomycin-Biosynthese statt, wenn zwei 3-Hydroxy-4-methylanthraniloyl-(4-MHA)-Pentapeptidlacton-Zwischenstufen oxidativ miteinander kondensiert werden. Enzymatische und genetische Arbeiten in Streptomyces chrysomallus haben die Schritte des Aufbaus der 4-MHA Pentapeptidlactone an den multifunktionellen Actinomycin Synthetasen aus den Pentapeptidlactonring-Aminosäuren und 4-MHA geklärt. Dagegen waren die Schritte der Biosynthese der 4-MHA nur teilweise auf enzymatischer Ebene charakterisiert und die verantwortlichen Gene unbekannt. Die Sequenzanalyse des Actinomycin-Biosynthese-Genclusters von S. chrysomallus führte zur Auffindung von 4-MHA Biosynthese Genen wie für Tryptophandioxygenase (acmG), Kynureninformamidase (acmF) und Hydroxykynureninase (acmH). Die betreffenden Enzymaktivitäten konnten durch vergleichende enzymatische Analysen von Zellextrakten des Actinomycin-produzierenden Wildtyp-Stamms und einer Actinomycin-negativen Mutante, in der diese Gene deletiert waren, identifiziert und von den entsprechenden katabolischen Enzymen des Primärstoffwechsels funktionell unterschieden werden. Weitere als Methyltransferase Gene annotierte Gene (acmI und acmL) wurden heterolog in E. coli exprimiert. Testung von AcmI und AcmL ergab, dass sie 3-Hydroxykynurenin (3-HK) zu 3-Hydroxy-4-methylkynurenin (4-MHK) aber nicht die 3-Hydroxyanthranilsäure (3-HA) zu 4-MHA - wie Daten früherer Arbeiten eigentlich voraussagten - methylieren. Demgemäß resultierte Zugabe von überschüssiger 3-HA zu S. chrysomallus und Streptomyces parvulus als auch zu Streptomyces antibioticus keine Stimulierung der Actinomycinsynthese. Stattdessen wurde die im Überschuss vorliegende 3-HA aufgrund ihrer strukturellen Homologie anstelle der intrazellulären 4-MHA in den Phenoxazinon-Chromophor neuer Actinomycine, der C-Demethylactinomycine, eingebaut. Darüber hinaus induzierte die 3-HA in S. antibioticus zusätzlich die Bildung von neuen C-Demethyl-hemi-Actinomycinen, die nur einen statt zwei Pentapeptidlactonringen am Phenoxazinonchromophor tragen. Entsprechend resultierte die Fütterung von S. antibioticus mit überschüssiger 4-MHA in der Bildung von Hemiactinomycinen. Letztere und die Demethyl-hemi-Actinomycine entstehen durch oxidative Kondensation im Überschuss präsenter 3-HA oder 4-MHA mit vorgebildeten Actinomycinhälften. Diese Reaktionen werden offensichtlich von der Phenoxazinon Synthase (PHS) in S. antibioticus katalysiert, die in S. chrysomallus oder S. parvulus nicht vorkommt. Weitere enzymatische und kinetische Charakterisierungen von AcmI und AcmL zeigten, dass sie neben ihrem eigentlichen Substrat, 3-Hydroxy-D-Kynurenin, auch andere phenolische Aminosäuren mit neuartiger antipodaler Stereospezifität am aromatischen Kern methylieren. Diese wird bestimmt von der optischen Konfiguration am alpha-C und gleichzeitig der unterschiedlichen Länge der aliphatischen Aminosäureseitenkette. Wildtyp AcmI/AcmL wurden auch in Zellextrakten von S. chrysomallus nachgewiesen, in welchen sie zusammen mit Hydroxykynureninase die Konversion von 3-HK (über 4-MHK) in 4-MHA katalysierten. Die in geringem Ausmaß stattfindende vorzeitige Spaltung von 3-HK in 3-HA durch Hydroxykynureninase ist Grund für das Vorkommen von Spuren von C-Demethylactinomycinen in natürlichen Actinomycingemischen.Actinomycins are a family of bicyclic chromopeptides with cytostatic activity produced by various streptomycetes. In their structures, two pentapeptide lactone rings are attached to a dimethyl-phenoxazinone-di-carboxylic acid in amide linkage. Biosynthetic studies in Streptomyces chrysomallus had shown earlier that phenoxazinone formation takes place during the last step of actinomycin synthesis by condensation of two 3-hydroxy-4-methylanthraniloyl (4-MHA) pentapeptide lactones (actinomycin half molecules). The 4-MHA pentapeptide lactones are assembled from 4-MHA and the amino acids of the pentapeptide lactone rings by a set of four non-ribosomal peptide synthetase subunits (actinomycin synthetases, ACMS). In contrast to actinomycin assembly, the steps of 4-MHA biosynthesis were less clear and the genes encoding these enzymes were as yet not known. Sequence analysis of DNA-regions flanking the NRPS genes of the actinomycin biosynthetic gene cluster from S. chrysomallus revealed genes encoding proteins with similarity to tryptophan dioxygenase, kynurenineformamidase, and hydroxykynureninase most probably involved in the biosynthesis of 4-MHA. Comparative enzymatic analysis of the corresponding enzyme activities in S. chrysomallus and the knockout mutant S. chrysomallus-white clearly distinguished these pathway specific enzymes from their homologues involved in cellular tryptophan catabolism and proved their involvement in 4-MHA synthesis. In addition, the gene cluster contained two paralogous genes, acmI and acmL, both encoding two nearly identical methyltransferases. Both genes were expressed in E. coli. Testing the resultant purified proteins revealed that each exclusively methylated 3-hydroxykynurenine (3-HK) with formation of 3-hydroxy-4-methylkynurenine (4-MHK) but not 3-hydroxyanthranilic acid (3-HA) to 3-hydroxy-4-methylanthranilic acid (4-MHA). Further analysis of these enzymes showed that they can methylate other phenolic amino acids, too, such as tyrosine but at a lower rate than 3-HK. The methylation reactions were stereospecific depending on the length of the amino acid side chain of these substrates resulting in exclusive methylation of 3-hydroxy-D-kynurenine (or homo-D-tyrosine) or of L-tyrosine (meta-L-tyrosine). Testing protein extracts of S. chrysomallus actively synthesizing actinomycins showed that they catalysed methylation of 3-HK whereas methylation of 3-HA was not observed. This proved that acmI and/or acmL were expressed during actinomycin synthesis. Moreover, external addition of 3-HA to actinomycin-producing S. chrysomallus, Streptomyces parvulus and Streptomyces antibioticus did not stimulate actinomycin synthesis but resulted in the formation of novel compounds, C-demethylactinomycins, due to incorporation of 3-HA into actinomycins instead of the structurally similar 4-MHA. This proved again, that 3-HK is precursor of 4-MHA rather than 3-HA. In addition, external addition of 3-HA and also of 4-MHA to S. antibioticus cultures led to the formation of as yet unknown novel actinomycin and demethylactinomycin homologues, hemiactinomycins, which contain only one pentapeptide lactone ring due to premature condensation of 3-HA or 4-MHA with actinomycin halves. Most probably the formation of these hemi-compounds is catalyzed by phenoxazinone-synthase (PHS) of S. antibioticus because S. chrysomallus or S. parvulus, both lacking PHS activity, do not produce hemiactinomycin and C-demethylactinomycin when fed with 3-HA or 4-MHA

The Actinomycin Biosynthetic Gene Cluster of Streptomyces chrysomallus: a Genetic Hall of Mirrors for Synthesis of a Molecule with Mirror Symmetry ▿

A gene cluster was identified which contains genes involved in the biosynthesis of actinomycin encompassing 50 kb of contiguous DNA on the chromosome of Streptomyces chrysomallus. It contains 28 genes with biosynthetic functions and is bordered on both sides by IS elements. Unprecedentedly, the cluster consists of two large inverted repeats of 11 and 13 genes, respectively, with four nonribosomal peptide synthetase genes in the middle. Nine genes in each repeat have counterparts in the other, in the same arrangement but in the opposite orientation, suggesting an inverse duplication of one of the arms during the evolution of the gene cluster. All of the genes appear to be organized into operons, each corresponding to a functional section of actinomycin biosynthesis, such as peptide assembly, regulation, resistance, and biosynthesis of the precursor of the actinomycin chromophore 4-methyl-3-hydroxyanthranilic acid (4-MHA). For 4-MHA synthesis, functional analysis revealed genes that encode pathway-specific isoforms of tryptophan dioxygenase, kynurenine formamidase, and hydroxykynureninase, which are distinct from the corresponding enzyme activities of cellular tryptophan catabolism in their regulation and in part in their substrate specificity. Phylogenetic analysis indicates that the pathway-specific tryptophan metabolism in Streptomyces most probably evolved divergently from the normal pathway of tryptophan catabolism to provide an extra or independent supply of building blocks for the synthesis of tryptophan-derived secondary metabolites

Genome Mining of <i>Micromonospora yangpuensis</i> DSM 45577 as a Producer of an Anthraquinone-Fused Enediyne

A new anthraquinone-fused enediyne, yangpumicin A (YPM A, <b>1</b>), along with four Bergman cyclization congeners (YPM B–E, <b>2</b>–<b>5</b>), was isolated from <i>Micromonospora yangpuensis</i> DSM 45577 after mining enediyne biosynthetic gene clusters from public actinobacterial genome databases and prioritizing the hits by an enediyne genome neighborhood network analysis for discovery. YPM A is potent against a broad spectrum of human cancer cell lines. The discovery of <b>1</b> provides new opportunities for the functionalization of enediynes to develop new conjugation chemistries for antibody–drug conjugates

PokMT1 from the Polyketomycin Biosynthetic Machinery of <i>Streptomyces diastatochromogenes</i> Tü6028 Belongs to the Emerging Family of <i>C</i>‑Methyltransferases That Act on CoA-Activated Aromatic Substrates

Recent biochemical characterizations of the MdpB2 CoA ligase and MdpB1 <i>C</i>-methyltransferase (<i>C</i>-MT) from the maduropeptin (MDP, <b>2</b>) biosynthetic machinery revealed unusual pathway logic involving C-methylation occurring on a CoA-activated aromatic substrate. Here we confirmed this pathway logic for the biosynthesis of polyketomycin (POK, <b>3</b>). Biochemical characterization unambiguously established that PokM3 and PokMT1 catalyze the sequential conversion of 6-methylsalicylic acid (6-MSA, <b>4</b>) to form 3,6-dimethylsalicylyl-CoA (3,6-DMSA-CoA, <b>6</b>), which serves as the direct precursor for the 3,6-dimethylsalicylic acid (3,6-DMSA) moiety in the biosynthesis of <b>3</b>. PokMT1 catalyzes the C-methylation of 6-methylsalicylyl-CoA (6-MSA-CoA, <b>5</b>) with a <i>k</i>_cat of 1.9 min^–1 and a <i>K</i>_m of 2.2 ± 0.1 μM, representing the most proficient <i>C</i>-MT characterized to date. Bioinformatics analysis of MTs from natural product biosynthetic machineries demonstrated that PokMT1 and MdpB1 belong to a phylogenetic clade of <i>C</i>-MTs that preferably act on aromatic acids. Significantly, this clade includes the structurally characterized enzyme SibL, which catalyzes C-methylation of 3-hydroxykynurenine in its free acid form, using two conserved tyrosine residues for catalysis. A homology model and site-directed mutagenesis suggested that PokMT1 also employs this unusual arrangement of tyrosine residues to coordinate C-methylation but revealed a large cavity capable of accommodating the CoA moiety tethered to <b>5</b>. CoA activation of the aromatic acid substrate may represent a general strategy that could be exploited to improve catalytic efficiency. This study sets the stage to further investigate and exploit the catalytic utility of this emerging family of <i>C</i>-MTs in biocatalysis and synthetic biology