Characterization of the Ketosynthase and Acyl Carrier Protein Domains at the LnmI Nonribosomal Peptide Synthetase–Polyketide Synthase Interface for Leinamycin Biosynthesis

Leinamycin (LNM) is biosynthesized by a hybrid nonribosomal peptide synthetase (NRPS)–acyltransferase (AT)-less type I polyketide synthase (PKS). Characterization of LnmI revealed ketosynthase (KS)–acyl carrier protein (ACP)–KS domains at the NRPS–PKS interface. Inactivation of the KS domain or ACP domain in vivo abolished LNM production, and the ACP domain can be phosphopantetheinylated in vitro. The LnmI KS–ACP–KS architecture represents a new mechanism for functional crosstalk between NRPS and AT-less type I PKS in the biosynthesis of hybrid peptide–polyketide natural products

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

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

Crystal Structure of Thioesterase SgcE10 Supporting Common Polyene Intermediates in 9- and 10-Membered Enediyne Core Biosynthesis

Enediynes are potent natural product anticancer antibiotics, and are classified as 9- or 10-membered according to the size of their enediyne core carbon skeleton. Both 9- and 10-membered enediyne cores are biosynthesized by the enediyne polyketide synthase (PKSE), thioesterase (TE), and PKSE-associated enzymes. Although the divergence between 9- and 10-membered enediyne core biosynthesis remains unclear, it has been observed that nascent polyketide intermediates, tethered to the acyl carrier protein (ACP) domain of PKSE, could be released by TE in the absence of the PKSE-associated enzymes. In this study, we determined the crystal structure of SgcE10, the TE that participates in the biosynthesis of the 9-membered enediyne C-1027. Structural comparison of SgcE10 with CalE7 and DynE7, two TEs that participate in the biosynthesis of the 10-membered enediynes calicheamicin and dynemicin, respectively, revealed that they share a common α/β hot-dog fold. The amino acids involved in both substrate binding and catalysis are conserved among SgcE10, CalE7, and DynE7. The volume and the shape of the substrate-binding channel and active site in SgcE10, CalE7, and DynE7 confirm that TEs from both 9- and 10-membered enediyne biosynthetic machineries bind the linear form of similar ACP-tethered polyene intermediates. Taken together, these findings further support the proposal that the divergence between 9- and 10-membered enediyne core biosynthesis occurs beyond PKSE and TE catalysis

Structure of the <i>ent</i>-Copalyl Diphosphate Synthase PtmT2 from <i>Streptomyces platensis</i> CB00739, a Bacterial Type II Diterpene Synthase

Terpenoids are the largest and most structurally diverse family of natural products found in nature, yet their presence in bacteria is underappreciated. The carbon skeletons of terpenoids are generated through carbocation-dependent cyclization cascades catalyzed by terpene synthases (TSs). Type I and type II TSs initiate cyclization via diphosphate ionization and protonation, respectively, and protein structures of both types are known. Most plant diterpene synthases (DTSs) possess three α-helical domains (αβγ), which are thought to have arisen from the fusion of discrete, ancestral bacterial type I TSs (α) and type II TSs (βγ). Type II DTSs of bacterial origin, of which there are no structurally characterized members, are a missing piece in the structural evolution of TSs. Here, we report the first crystal structure of a type II DTS from bacteria. PtmT2 from <i>Streptomyces platensis</i> CB00739 was verified as an <i>ent</i>-copalyl diphosphate synthase involved in the biosynthesis of platensimycin and platencin. The crystal structure of PtmT2 was solved at a resolution of 1.80 Å, and docking studies suggest the catalytically active conformation of geranylgeranyl diphosphate (GGPP). Site-directed mutagenesis confirmed residues involved in binding the diphosphate moiety of GGPP and identified DxxxxE as a potential Mg²⁺-binding motif for type II DTSs of bacterial origin. Finally, both the shape and physicochemical properties of the active sites are responsible for determining specific catalytic outcomes of TSs. The structure of PtmT2 fundamentally advances the knowledge of bacterial TSs, their mechanisms, and their role in the evolution of TSs

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 (<i>S</i>)-3-chloro-5-hydroxy-β-tyrosine. In a convergent biosynthesis of the C-1027 chromophore, the (<i>S</i>)-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 (<i>S</i>)-3-chloro-5-hydroxy-β-tyrosine and (<i>R</i>)-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 <i>R</i>-enantiomer of the acceptor substrate, accounting for the regio- and stereospecific ester-bond formation between SgcC2-tethered (<i>S</i>)-3-chloro-5-hydroxy-β-tyrosine and (<i>R</i>)-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

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 <i>Streptomyces globisporus</i>

C-1027 is a chromoprotein enediyne antitumor antibiotic produced by <i>Streptomyces globisporus</i>. In the last step of biosynthesis of the (<i>S</i>)-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 (<i>S</i>)-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