Shumpei Asamizu

Crystal structures and catalytic mechanism of cytochrome P450 StaP that produces the indolocarbazole skeleton

Staurosporine isolated from Streptomyces sp. TP-A0274 is a member of the family of indolocarbazole alkaloids that exhibit strong antitumor activity. A key step in staurosporine biosynthesis is the formation of the indolocarbazole core by intramolecular C–C bond formation and oxidative decarboxylation of chromopyrrolic acid (CPA) catalyzed by cytochrome P450 StaP (StaP, CYP245A1). In this study, we report x-ray crystal structures of CPA-bound and -free forms of StaP. Upon substrate binding, StaP adopts a more ordered conformation, and conformational rearrangements of residues in the active site are also observed. Hydrogen-bonding interactions of two carboxyl groups and T-shaped π–π interactions with indole rings hold the substrate in the substrate-binding cavity with a conformation perpendicular to the heme plane. Based on the crystal structure of StaP–CPA complex, we propose that C–C bond formation occurs through an indole cation radical intermediate that is equivalent to cytochrome c peroxidase compound I [Sivaraja M, Goodin DB, Smith M, Hoffman BM (1989) Science 245:738–740]. The subsequent oxidative decarboxylation reaction is also discussed based on the crystal structure. Our crystallographic study shows the first crystal structures of enzymes involved in formation of the indolocarbazole core and provides valuable insights into the process of staurosporine biosynthesis, combinatorial biosynthesis of indolocarbazoles, and the diversity of cytochrome P450 chemistry.

Theoretical and Experimental Studies of the Conversion of Chromopyrrolic Acid to an Antitumor Derivative by Cytochrome P450 StaP: The Catalytic Role of Water Molecules

Chromopyrrolic acid (CPA) oxidation by cytochrome P450 StaP is a key process in the biosynthesis of antitumor drugs (Onaka, H.; Taniguchi, S.; Igarashi, Y.; Furumai, T. Biosci. Biotechnol. Biochem. 2003, 67, 127-138), which proceeds by an unusual C-C bond coupling. Additionally, because CPA is immobilized by a hydrogen-bonding array, it is prohibited from undergoing direct reaction with Compound I, the active species of P450. As such, the mechanism of P450 StaP poses a puzzle. In the present Article, we resolve this puzzle by combination of theory, using QM/MM calculations, and experiment, using crystallography and reactivity studies. Theory shows that the hydrogen-bonding machinery of the pocket deprotonates the carboxylic acid groups of CPA, while the nearby His(250) residue and the crystal waters, Wat(644) and Wat(789), assist the doubly deprotonated CPA to transfer electron density to Compound I; hence, CPA is activated toward proton-coupled electron transfer that sets the entire mechanism in motion. The ensuing mechanism involves a step of C-C bond formation coupled to a second electron transfer, four proton-transfer and tautomerization steps, and four steps where Wat(644) and Wat(789) move about and mediate these events. Experiments with the dichlorinated substrate, CCA, which expels Wat(644), show that the enzyme loses its activity. H250A and H250F mutations of P450 StaP show that His(250) is important, but in its absence Wat(644) and Wat(789) form a hydrogen-bonding diad that mediates the transformation. Thus, the water diad emerges as the minimal requisite element that endows StaP with function. This highlights the role of water molecules as biological catalysts that transform a P450 to a peroxidase-type (Derat, E.; Shaik, S. J. Am. Chem. Soc. 2006, 128, 13940-13949).

Crystal Structure of VioE, a Key Player in the Construction of the Molecular Skeleton of Violacein

Violacein and the indolocarbazoles are naturally occurring bisindole products with various biological activities, including antitumor activity. Although these compounds have markedly different molecular skeletons, their biosynthetic pathways share the same intermediate “compound X,” which is produced from l-tryptophan via indole-3-pyruvic acid imine. Compound X is a short-lived intermediate that is spontaneously converted to chromopyrrolic acid for indolocarbazole biosynthesis, whereas VioE transforms compound X into protodeoxyviolaceinic acid, which is further modified by other enzymes to produce violacein. Thus, VioE plays a key role in the construction of the molecular skeleton of violacein. Here, we present the crystal structure of VioE, which consists of two subunits, each of which forms a structure resembling a baseball glove. Each subunit has a positively charged pocket at the center of the concave surface of the structure. Mutagenesis analysis of the surface pocket and other surface residues showed that the surface pocket serves as an active site. We have also solved the crystal structure of a complex of VioE and phenylpyruvic acid as an analogue of a VioE-substrate complex. A docking simulation with VioE and the IPA imine dimer, which is proposed to be compound X, agreed with the results from the mutational analysis and the VioE-phenylpyruvic acid complex structure. Based on these results, we propose that VioE traps the highly reactive substrate within the surface pocket to suppress CPA formation and promote protodeoxyviolaceinic acid formation caused by proximity and orientation effects.

Evolutionary Divergence of Sedoheptulose 7-phosphate Cyclases Leads to Several Distinct Cyclic Products

Sedoheptulose 7-phosphate cyclases are enzymes that utilize the pentose phosphate pathway intermediate, sedoheptulose 7-phosphate, to generate cyclic precursors of many bioactive natural products, such as the antidiabetic drug acarbose, the crop protectant validamycin, and the natural sunscreens mycosporine-like amino acids. These proteins are phylogenetically related to the dehydroquinate (DHQ) synthases from the shikimate pathway and are part of the more recently recognized superfamily of sugar phosphate cyclases, which includes DHQ synthases, aminoDHQ synthases, and 2-deoxy-scyllo-inosose synthases. Through genome mining and biochemical studies, we identified yet another subset of DHQS-like proteins in the actinomycete Actinosynnema mirum and the myxobacterium Stigmatella aurantiaca DW4/3-1. These enzymes catalyze the conversion of sedoheptulose 7-phosphate to 2-epi-valiolone, which is predicted to be an alternative precursor for aminocyclitol biosynthesis. Comparative bioinformatics and biochemical analyses of these proteins with 2-epi-5-epi-valiolone synthases (EEVS) and desmethyl-4-deoxygadusol synthases (DDGS) provided further insights into their genetic diversity, conserved amino acid sequences, and plausible catalytic mechanisms. The results further highlight the uniquely diverse DHQS-like sugar phosphate cyclases, which may provide new tools for chemoenzymatic, stereospecific synthesis of various cyclic molecules.

5-Alkyl-1,2,3,4-tetrahydroquinolines, New Membrane-Interacting Lipophilic Metabolites Produced by Combined Culture of Streptomyces nigrescens and Tsukamurella pulmonis

Eight novel 5-alkyl-1,2,3,4-tetrahydroquinolines (5aTHQs) bearing different side chains have been isolated from a combined culture of Streptomyces nigrescens HEK616 and Tsukamurella pulmonis TP-B0596. The chemical structures including the absolute configuration were elucidated by spectroscopic analysis and total synthesis. 5aTHQs inhibited the growth of wild-type fission yeast while only weakly inhibiting the growth of several mutant strains synthesizing premature ergosterol. These results demonstrate that 5aTHQs are novel antifungals that may target cell membranes.

Insights into the biosynthesis of dehydroalanines in goadsporin

Dehydroalanines in goadsporin are proposed to be formed by GodF and GodG, which show slight homology to the N‐terminal glutamylation and C‐terminal elimination domains, respectively, of LanB, a class I lanthipeptide dehydratase. Although similar, separated‐type LanBs are conserved among thiopeptides and indispensable for their biosynthesis and biological activities, these enzymes had not yet been characterized. Here, we identified goadsporin B, which has unmodified Ser4 and Ser14, from both godF and godG disruptants. The godG disruptant also produced goadsporin C, a glutamylated‐Ser4 variant of goadsporin B. These results suggested that dehydroalanines are formed by glutamylation and glutamate elimination. NMR analysis revealed for the first time that the glutamyl group was attached to a serine via an ester bond, by the catalysis of LanB‐type enzymes. Our findings provide insights into the function of separated‐type LanBs involved in the biosynthesis of goadsporin and thiopeptides.

Genetic approaches to generate hyper-producing strains of goadsporin: the relationships between productivity and gene duplication in secondary metabolite biosynthesis

Improving the productivity of secondary metabolites is highly beneficial for the utilization of natural products. Here, we found that gene duplication of the goadsporin biosynthetic gene locus resulted in hyper-production. Goadsporin is a linear azole containing peptide that is biosynthesized via a ribosome-mediated pathway in Streptomyces sp. TP-A0584. Recombinant strains containing duplicated or triplicated goadsporin biosynthetic gene clusters produced 1.46- and 2.25-fold more goadsporin than the wild-type strain. In a surrogate host, Streptomyces lividans, chromosomal integration of one or two copies of the gene cluster led to 342.7 and 593.5 mg/L of goadsporin production. Expression of godI, a self-resistance gene, and of godR, a pathway-specific transcriptional regulator, under a constitutive promoter gave 0.79- and 2.12-fold higher goadsporin production than the wild-type strain. Our experiments indicated that a proportional relationship exists between goadsporin production per culture volume and the copy number of the biosynthetic gene cluster. Graphical Abstract Proportional relationship exists between Streptomyces antibiotic production and the copy number of the biosynthetic gene cluster.

Structure of a Sedoheptulose 7-Phosphate Cyclase: ValA from Streptomyces hygroscopicus

Sedoheptulose 7-phosphate cyclases (SH7PCs) encompass three enzymes involved in producing the core cyclitol structures of pseudoglycosides and similar bioactive natural products. One such enzyme is ValA from Streptomyces hygroscopicus subsp. jinggangensis 5008, which makes 2-epi-5-epi-valiolone as part of the biosynthesis of the agricultural antifungal agent validamycin A. We present, as the first SH7PC structure, the 2.1 Å resolution crystal structure of ValA in complex with NAD+ and Zn2+ cofactors. ValA has a fold and active site organization resembling those of the sugar phosphate cyclase dehydroquinate synthase (DHQS) and contains two notable, previously unrecognized interactions between NAD+ and Asp side chains conserved in all sugar phosphate cyclases that may influence catalysis. Because the domains of ValA adopt a nearly closed conformation even though no sugar substrate is present, comparisons with a ligand-bound DHQS provide a model for aspects of substrate binding. One striking active site difference is a loop that adopts a distinct conformation as a result of an Asp → Asn change with respect to DHQS and alters the identity and orientation of a key Arg residue. This and other active site differences in ValA are mostly localized to areas where the ValA substrate differs from that of DHQS. Sequence comparisons with a second SH7PC making a product with distinct stereochemistry lead us to postulate that the product stereochemistry of a given SH7PC is not the result of events taking place during catalysis but is accomplished by selective binding of either the α or β pyranose anomer of the substrate.

Mycolic acid-containing bacteria activate heterologous secondary metabolite expression in Streptomyces lividans

Mycolic acid-containing bacteria activate heterologous secondary metabolite expression in Streptomyces lividans

Evolution and Distribution of C7–Cyclitol Synthases in Prokaryotes and Eukaryotes

2-Epi-5-epi-valiolone synthase (EEVS), a C7-sugar phosphate cyclase (SPC) homologous to 3-dehydroquinate synthase (DHQS), was discovered during studies of the biosynthesis of the C7N-aminocyclitol family of natural products. EEVS was originally thought to be present only in certain actinomycetes, but analyses of genome sequences showed that it is broadly distributed in both prokaryotes and eukaryotes, including vertebrates. Another SPC, desmethyl-4-deoxygadusol synthase (DDGS), was later discovered as being involved in the biosynthesis of mycosporine-like amino acid sunscreen compounds. Current database annotations are quite unreliable, with many EEVSs reported as DHQS, and most DDGSs reported as EEVS, DHQS, or simply hypothetical proteins. Here, we identify sequence features useful for distinguishing these enzymes, report a crystal structure of a representative DDGS showing the high similarity of the EEVS and DDGS enzymes, identify notable active site differences, and demonstrate the importance of two of these active site residues for catalysis by point mutations. Further, we functionally characterized two representatives of a distinct clade equidistant from known EEVS and known DDGS groups and show them to be authentic EEVSs. Moreover, we document and discuss the distribution of genes that encode EEVS and DDGS in various prokaryotes and eukaryotes, including pathogenic bacteria, plant symbionts, nitrogen-fixing bacteria, myxobacteria, cyanobacteria, fungi, stramenopiles, and animals, suggesting their broad potential biological roles in nature.