片山 欣哉

Vicenistatin, an antitumor antibiotic isolated from Streptomyces halstedii HC34, is a unique 20-membered macrocyclic lactam with a novel aminosugar vicenisamine. We have been interested in the biosynthesis of vicenistatin, because the aglycon is distinct from regular polyketides solely consisting of lower fatty acid units. Isotope tracer experiments of ^<13>C-labeled acetate and propionate showed that C-1 to C-16 of the aglycon was derived from acetate and propionate in a standard polyketide biosynthetic pathway, and incorporation of intact acetate into C-17 and C-18 strongly suggested that a possible starter unit was derived from glutamate via 3-methylaspartate. Feeding experiments of deuterated glutamate, [^<15>N]-glutamate and deuterated (2S,3S)-3-methylaspartate showed that glutamate mutase, which is known to convert glutamate to 3-methylaspartate, was actually involved in the biosynthesis of the aglycon. However, (2S,3R)-3-methylaspartate and DL-3-amino-2-methylpropionate were not incorporated into vicenistatin. Thus, epimerization of the methylated site is involved in the starter biosynthesis, but its timing is yet to be clarified. The vicenistatin gene cluster (vin) spanning ca. 64kbp was successfully cloned and sequenced by using consensus sequences of dTDP-glucose 4,6-dehydratase and dTDP-4-keto-6-deoxyglucose 2,3-dehydratase which were usually involved in the early steps of the 2,6-deoxysugar biosynthesis. The vin cluster contains ORFs encoding proteins homologous to polyketide synthases (vinP1-4), glutamate mutase (vinH, I), acyl CoA ligase (vinN) and decarboxylase (vinO). VinN and VinO appear to be involved in the formation of the starter unit of aglycon from the earlier intermediate 3-methylaspartate generated by VinH and VinI. VinP1-4, consisting of eight extending modules and a terminal thioesterase domain, are responsible to produce the whole aglycon polyketide. Also contained in the cluster are ORFs homologous to 4,6-dehydratase (vinB), 2,3-dehydratase (vinD), aminotransferase (vinF), N-methyltransferase (vinG) and glycosyltransferase (vinC), for the vicenisamine biosynthesis. Furthermore, VinC was heterologously expressed in Escherichia coil and purified. The glycosyl transfer reaction from dTDP-vicenisamine to the aglycon was confirmed with the recombinant VinC. These results proved that the abovementioned gene cluster encodes the vicenistatin biosynthetic enzymes.

Mutations in the Streptomyces peucetius dnrD gene block the ring cyclization leading from aklanonic acid methyl ester to aklaviketone, an intermediate in the biosynthetic pathway to daunorubicin and doxorubicin. To investigate the role of DnrD in this transformation its gene was overexpressed in Escherichia coli and the DnrD protein was purified to homogeneity and characterized. The enzyme was shown to catalyse the conversion of aklanonic acid methyl ester to aklaviketone presumably via an intramolecular aldol condensation mechanism. In contrast to the analogous intramolecular aldol cyclization catalyzed by the TcmI protein from the tetracenomycin C pathway in Streptomyces glaucescens, where a tricyclic anthraquinol carboxylic acid is converted to its fully aromatic tetracyclic form, the conversion catalyzed by DnrD occurs after anthraquinone formation and requires activation of a carboxylic acid group by esterification of aklanonic acid, the aklanonic acid methyl ester precursor. Also, the cyclization is not coupled with a subsequent dehydration step that would result in an aromatic ring. As the substrates for the DnrD and TcmI enzymes are among the earliest isolable intermediates of aromatic polyketide biosynthesis, an understanding of the mechanism and active site topology of these proteins will allow one to determine the substrate and mechanistic parameters important for aromatic ring formation. In the future, these parameters may be able to be applied to some of the earlier polyketide cyclization processes that currently are difficult to study in vitro.

Solanapyrone synthase has been partially purified from a phytopathogenic fungus Alternaria solani. On the basis of the chromatographic behavior, it is speculated that single enzyme catalyzes both oxidation of 6 and the subsequent Diels-Alder reaction of 7. The enzyme is monomeric and showed native molecular weight 40 kD and pl 4.25. The substrate specificity of this enzyme was also examined with various substrate analogs. Enzymatic Diels-Alder reaction of 6 in semi-preparative scale afforded (-)-1 with high enantioselectivity and with good exo-selectivity which is difficult to attain by che...

First enzyme which catalyzes Diels-Alder reaction has been partially purified. This crude enzyme is able to catalyze [4+2]-cycloaddition of prosolanapyrone III to the exo adduct solanapyrone A and the endo adduct solanapyrone D in a 7:1 ratio differing substantially from the ratio found in a background reaction (exo/endo, 1:23). The optical purity of the enzymatic reaction product solanapyrone A was estimated as 92%ee by HPLC analysis monitored with CD spectrometer. The enzyme also exhibited an oxidase activity of prosolanapyrone II to III. Two-step conversion of prosolanapyrone II to solan...

Involvement of Diels-Alder reaction in biosynthesis of secondary metabolites has been proposed in many instances. However the presence of the enzyme responsible for this biological unusual reaction has never been proved. In order to examine this possibility, we choose a phytotoxin, solanapyrone A (1) whose skeleton was presumably constructed via intramolecular Diels-Alder reaction. Here, we describe results of successful incorporation of precursors (7a) and (7b) prior to [4+2]cycloaddition. The specification of precursor of [4+2]cycloaddition were made as follows. From the incorporation experiments of [S-CD_3]methionine and [^2H]-labeled 8 suggested that oxidations of methyl group at C-14 occurs prior to [4+2] cycloaddition. Since polyenepyrones such as citreomontanine (6) are frequently occurred in natural products, we speculate that formation of pyrone ring and introduction of two C1-units take place before cyclization and that trienes (5) and (7) must be actual intermediates. The deuterium labeled plausible precursors (7a) and (7b) of Diels-Alder reaction were synthesized and incorporated. In ^2H-NMR spectra of 1 and 2 obtained from feeding experiments, the signals originated from OCH_3 and CHO and/or CH_2OH were observed. These data clearly indicated that less oxidized precursor, triene (7) was oxidized to aldehyde (5) which was subsequently cyclized to give solanapyrone A (1).