Makoto Hashimoto

A plethora of dimeric natural products exist with diverse chemical structures and biological activities. A major strategy for dimerization is aryl coupling reactions catalyzed by cytochrome P450 or laccase. Actinorhodin (ACT) from Streptomyces coelicolor has a dimeric pyranonaphthoquinone structure connected by a C-C bond. Here, we identified a NmrA-family dimerizing enzyme, ActVA-ORF4, and a cofactor independent oxidase, ActVA-ORF3, both involved in the last step of ACT biosynthesis. ActVA-ORF4 is a unique  NAD(P)H-dependent enzyme that catalyzes the inter-molecular C-C bond formation using 8-hydroxydihydrokalafungin (DHK-OH) as the sole substrate. On the other hand, ActVA-ORF3 was found to be a quinone-forming enzyme that produces the coupling substrate, DHK-OH, and the final product, ACT. Consequently, the functional assignment of all essential enzymes in ACT biosynthesis was completed, which would be a landmark in our understanding of the entire biosynthetic pathway for one of the best-known model natural products, ACT.

Actinorhodin (ACT) is a benzoisochromanequinone antibiotic produced by Streptomyces coelicolor A3(2), which has served as a favored model organism for comprehensive studies of antibiotic biosynthesis and its regulation. (S)-DNPA undergoes various modifications as an intermediate in the ACT biosynthetic pathway, including enoyl reduction to DDHK. It has been suggested that actVI-ORF2 encodes an enoyl reductase (ER). However, its function has not been characterized in vitro. In this study, biochemical analysis of recombinant ActVI-ORF2 revealed that (S)-DNPA is converted to DDHK in a stereospecific manner with NADPH acting as a cofactor. (R)-DNPA was also reduced to 3-epi-DDHK with the comparable efficacy as (S)-DNPA, suggesting that the stereospecificity of ActVI-ORF2 was not affected by the stereochemistry at the C-3 of DNPA. ActVI-ORF2 is a new example of a discrete ER, which is distantly related to known ERs according to phylogenetic analysis.

Shimalactones A and B are neuritogenic polyketides possessing characteristic oxabicyclo[2.2.1]heptane and bicyclo[4.2.0]octadiene ring systems that are produced by the marine fungus Emericella variecolor GF10. We identified a candidate biosynthetic gene cluster and conducted heterologous expression analysis. Expression of ShmA polyketide synthase in Aspergillus oryzae resulted in the production of preshimalactone. Aspergillus oryzae and Saccharomyces cerevisiae transformants expressing ShmA and ShmB produced shimalactones A and B, thus suggesting that the double bicyclo-ring formation reactions proceed non-enzymatically from preshimalactone epoxide. DFT calculations strongly support the idea that oxabicyclo-ring formation and 8π-6π electrocyclization proceed spontaneously after opening of the preshimalactone epoxide ring through protonation. We confirmed the formation of preshimalactone epoxide in vitro, followed by its non-enzymatic conversion to shimalactones in the dark.

The asteltoxin-producing fungus Emericella variecolor IFM42010 possesses 22 highly-reducing polyketide synthase (HR-PKS) genes. Of these, an HR-PKS with a methyltransferase domain but lacking an enoylreductase domain could be involved in the biosynthesis of asteltoxin and related compounds. From six such candidate HR-PKS genes, Ev460pks was analyzed by gene disruption in E. variecolor and heterologous expression in Aspergillus oryzae. The Ev460pks-disrupted strain retained asteltoxin production ability, indicating that Ev460pks is not involved in asteltoxin biosynthesis. The A. oryzae transformant harboring the Ev460pks gene produced compounds 1 and 2, along with several unidentified products possibly decomposed from 2. Spectroscopic analyses revealed that 1 was a 4-methyl-β-ketolactone with a methylheptatriene side-chain at the C-5 position, and 2 was also a 4-methyl-β-ketolactone, bearing a dimethyltetradecahexaene side-chain at the same position. The relative configuration at C-4 in compounds 1 and 2 was opposite.

CsyB from Aspergillus oryzae is a novel type III polyketide synthase that catalyzes the formation of csypyrone B1 [4-(3-acetyl-4-hydroxy-2-oxo-2H-pyran-6- yl)butyric acid] from fatty acyl-CoA, malonyl-CoA and acetoacetyl-CoA. Recombinant CsyB expressed in Escherichia coli was crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space P21, with unit-cell parameters a = 70.0, b = 104.8, c = 73.5 Å, β = 114.4°. © 2014 International Union of Crystallography All rights reserved.

Pamamycin (PM, 1) is an aerial mycelium inducer isolated from Streptomyces alboniger. It is a unique sixteen-membered macrodiolide with a dimethylamino group-bearing side chain and dimethylamino group of PM is indispensable for the activity. Feeding experiments with <13>^C-labeled acetate and propionate, or <15>^N-labeled precursor showed that the carbon skeleton of PM-607 (MW 607) was assembled from six acetate, four propionate and three succinate units, and the nitrogen atom was derived from α-amino group of amino acid. We also revealed that two N-methyl groups of PM were derived from methionine by feeding experiment with [methyl-2^H_3]-methionine. Analyses of these intermediates accumulated in the blocked mutants of PM biosynthesis showed accumulation of hydroxy acids 3, 4 and 5, and the absence of ketone intermediate 2. These results indicated that the transamination occurred in keto-hydroxy acid 3 to generate 5. Quantitative analyses of PM intermediates 3, 4, 5 and PM in the cultured materials of parent strain indicated that transamination was the rate-determining step in PM production.