Carbon-free production of 2-deoxy-scyllo-inosose (DOI) in cyanobacterium Synechococcus elongatus PCC 7942.

Owing to their photosynthetic capabilities, there is increasing interest in utilizing cyanobacteria to convert solar energy into biomass. 2-Deoxy-scyllo-inosose (DOI) is a valuable starting material for the benzene-free synthesis of catechol and other benzenoids. DOI synthase (DOIS) is responsible for the formation of DOI from d-glucose-6-phosphate (G6P) in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics such as neomycin and butirosin. DOI fermentation using a recombinant Escherichia coli strain has been reported, although a carbon source is necessary for high-yield DOI production. We constructed DOI-producing cyanobacteria toward carbon-free and sustainable DOI production. A DOIS gene derived from the butirosin producer strain Bacillus circulans (btrC) was introduced and expressed in the cyanobacterium Synechococcus elongatus PCC 7942. We ultimately succeeded in producing 400 mg/L of DOI in S. elongatus without using a carbon source. DOI production by cyanobacteria represents a novel and efficient approach for producing benzenoids from G6P synthesized by photosynthesis.

Biosynthesis of 2-deoxystreptamine-containing antibiotics in Streptoalloteichus hindustanus JCM 3268: characterization of 2-deoxy-scyllo-inosose synthase.

A part of the new biosynthetic gene cluster for 2-deoxystreptamine-containing antibiotics was identified from Streptoalloteichus hindustanus. The alloH gene in the gene cluster was deduced to encode 2-deoxy-scyllo-inosose synthase and the expressed protein AlloH was confirmed to have this enzyme activity. Furthermore, biochemical properties of AlloH were studied.

Inhibition of type 2 isopentenyl diphosphate isomerase from Methanocaldococcus jannaschii by a mechanism-based inhibitor of type 1 isopentenyl diphosphate isomerase.

Type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2, EC 5.3.3.2) is a flavoprotein, which requires FMN, NADPH, and Mg2+ for the activity to convert isopentenyl diphosphate to dimethylallyl diphosphate. For investigation of the reaction mechanism of IDI-2, 3,4-epoxy-3-methylbutyl diphosphate (EIPP), a mechanism-based inhibitor of type 1 IDI (IDI-1), was treated with the overexpressed IDI-2 (MjIDI) from methanogenic archaeon Methanocaldococcus jannaschii. EIPP showed the time- and concentration-dependent inhibition (KI; 56.5 mM, k(inact); 0.10 s(-1), k(inact)/KI; 1.76 s(-1)M(-1)) and the UV-vis spectrum of MjIDI after treatment with EIPP was apparently different from that of the untreated MjIDI. These results indicated that EIPP modified FMN through a covalent bond in the active site of MjIDI. The formed EIPP-FMN complex was separated from the reaction mixture and the spectrometric analysis of the complex suggested that the reduced form of FMN bound to EIPP at the N5 position. These results may suggest that the IDI-2 reaction is similar to IDI-1, which proceeds via carbocation-type intermediate.

The complete biosynthetic gene cluster of the 28-membered polyketide macrolactones, halstoctacosanolides, from Streptomyces halstedii HC34.

Halstoctacoanolides A and B are 28-membered polyketide macrolactones and were isolated from Streptomyces halstedii HC34. The biosynthetic gene cluster (hls cluster) of halstoctacosanolides was completely identified from the genome library of Streptomyces halstedii HC34. DNA sequence analysis of ca. 100 kb region revealed that there were seven type I polyketide synthases (PKSs) and two cytochrome P450 monooxygenases in this cluster. Involvement of the gene cluster in the halstoctacosanolide biosynthesis was demonstrated by the gene disruption of P450 monooxygenase genes. The mutants produced a new deoxygenated halstoctacosanolide derivative, halstoctacosanolide C, which confirmed that the hls gene cluster was essential for the biosynthesis of halstoctacosanolides.

Involvement of glutamate mutase in the biosynthesis of the unique starter unit of the macrolactam polyketide antibiotic vicenistatin.

The macrolactam antibiotic vicenistatin, produced in Streptomyces halstedii HC34, is biosynthesized by the polyketide pathway, using a unique 3-methylaspartate-derived molecule as starter unit. The vinI gene in the vicenistatin biosynthetic gene cluster encoding glutamate mutase, which rearranges glutamate to 3-methylaspartate, was disrupted. The vinI disruption completely abolished the production of vicenistatin, while the disruptant recovered the production of vicenistatin when 3-methylaspartate was added to the culture. These results indicate that vinI is essential for the 3-methylaspartate formation in the vicenistatin biosynthesis. Furthermore, the mutant accumulated new vicenistatin derivatives (desmethylvicenistatins), which lacked a methyl group in the starter unit. The desmethylvicenistatins were shown by feeding experiments to be derived from aspartate instead of 3-methylaspartate as the starter unit. These results indicate that the vicenistatin polyketide synthase can accept alternative starter units toward the production of novel polyketides.

Extended sequence and functional analysis of the butirosin biosynthetic gene cluster in Bacillus circulans SANK 72073.

Butirosin produced by Bacillus circulans is among the clinically important 2-deoxystreptamine containing aminoglycoside antibiotics and its unique structure is found in (S)-4-amino-2-hydroxyburyric acid substituted at C-1 of 2-deoxystreptamine. Recently, the key part of the butirosin biosynthetic gene cluster has been identified from Bacillus circulans SANK 72073, however the whole gene for the biosynthesis awaited for identification. In the present study, we undertook extended analysis of the butirosin biosynthetic gene cluster and found nine additional open reading flames (ORFs), btrQ, btrR1, btrR2, btrT, btrU, btrV, btrW, btrX and orf1 in the cluster. In addition, we constructed disruption mutants of btrR1 and btrP-V, and found that the btr genes (ca. 24Kb) between btrR1 and btrP-V are at least required for the butirosin biosynthesis.

Stereochemical recognition of doubly functional aminotransferase in 2-deoxystreptamine biosynthesis.

The doubly functional aminotransferase BtrS in the 2-deoxystreptamine (DOS) biosynthesis, in which two transaminations are involved, was characterized by a genetic as well as a chemical approach with the heterologously expressed enzyme. The gene disruption study clearly showed that BtrS is involved, in addition to the previously confirmed first transamination, in the second transamination as well. This dual function of BtrS for the DOS biosynthesis was further confirmed by the structural determination of the reverse reaction product from DOS. Enantiospecific formation of the reverse reaction product from DOS clearly showed that BtrS distinguishes the enantiotopic amino groups of DOS, but in contrast, both enantiomers of 2-deoxy-scyllo-inosose (DOI) were efficiently accepted by BtrS to give a racemic product. This unique stereochemical recognition of DOI chirality and DOS prochirality by BtrS is mechanistically explained by a specific hydrogen-bond donating force in the enzyme active site as a particular feature of this doubly functional enzyme.

Enzymatic approach to unnatural glycosides with diverse aglycon scaffolds using glycosyltransferase VinC.

Glycosyltransferase VinC was explored for a construction of glycoside libraries using dTDP-vicenisamine and structurally unrelated unnatural aglycons, and new unnatural vicenisaminides were successfully constructed. Structural elements of aglycon recognition by VinC were proposed by modeling studies and were confirmed by the success of transglycosylation upon a designed aglycon.

Stereospecificity of hydride transfer in NAD+-catalyzed 2-deoxy-scyllo-inosose synthase, the key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics.

The key enzyme in the biosynthesis of clinically important aminocyclitol antibiotics is 2-deoxy-scyllo-inosose synthase (DOIS), which converts ubiquitous d-glucose 6-phosphate (G-6-P) into the specific carbocycle, 2-deoxy-scyllo-inosose with an aid of NAD(+)-NADH recycling. The NAD(+)-dependent first step of the DOIS reaction was examined in detail by the use of 6-phosphonate and 6-homophosphonate analogs of G-6-P. Both analogs showed competitive inhibition against the DOIS reaction with K(i) values of 1.3 and 2.8 mM, respectively, due to their inability for the subsequent phosphate elimination. Based on the direct spectrophotometric observation of NADH formed by the hydride transfer from 6-phosphonate to NAD(+), the stereospecificity of the hydride transfer in the DOIS reaction was analyzed with 6-[4-(2)H]phosphonate and was found to be pro-R specific.

A new family of glucose-1-phosphate/glucosamine-1-phosphate nucleotidylyltransferase in the biosynthetic pathways for antibiotics.

Aminoglycoside antibiotics are composed of aminosugars and a unique aminocyclitol aglycon including 2-deoxystreptamine (DOS), streptidine, actinamine, etc., and nucleotidylyltransferases, sugar modifying enzymes, and glycosyltransferases appear to be essential for their biosynthesis. However, the genes encoding those enzymes were unable to be identified by a standard homology search in the butirosin biosynthetic btr gene cluster, except that the btrM gene appeared to be a glycosyltransfease. Disruption studies of the btrD gene indicated that BtrD was involved in the supply of a glycosyl donor immediately prior to the glycosylation of DOS giving paromamine. As anticipated, BtrD expressed in Escherichia coli was able to catalyze UDP-D-glucosamine formation from D-glucosamine-1-phosphate and UTP. Both dTTP and UTP were good NTP substrates, and D-glucose-1-phosphate and D-glucosamine-1-phosphate were good sugar phosphates for the enzyme reaction. This finding is the first to identify an enzyme which activates a sugar donor in the DOS-containing antibiotics. Interestingly, BtrD homologues have been reported as functionally unknown open reading frames (ORFs) in the biosynthetic gene clusters for several antibiotics including teicoplanin, balhimycin, chloroeremomycin, and mitomycin C. It appears therefore that gene clusters for antibiotic biosynthesis provide their own nucleotidylyltransferases, and the BtrD homologues are among the secondary metabolism specific enzymes.

Biosynthesis of 2-deoxystreptamine by three crucial enzymes in Streptomyces fradiae NBRC 12773.

NeoA, B, and C encoded in the neomycin biosynthetic gene cluster have been enzymatically confirmed to be responsible to the formation of 2-deoxystreptamine (DOS) in Streptomyces fradiae. NeoC was functionally characterized as 2-deoxy-scyllo-inosose synthase, which catalyzes the carbocycle formation from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose. Further, NeoA appeared to catalyze the oxidation of 2-deoxy-scyllo-inosamine (DOIA) with NAD(P)+ forming 3-amino-2,3-dideoxy-scyllo-inosose (amino-DOI). Consequently, NeoA was characterized as 2-deoxy-scyllo-inosamine dehydrogenase. Finally, amino-DOI produced by NeoA from DOIA was transformed into DOS by NeoB. Since NeoB (Neo6) was also reported to be L-glutamine:2-deoxy-scyllo-inosose aminotransferase, all the enzymes in the DOS biosynthesis were characterized for the first time.

Crystallization and X-ray analysis of 2-deoxy-scyllo-inosose synthase, the key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics.

A recombinant 2-deoxy-scyllo-inosose synthase from Bacillus circulans has been crystallized at 277 K using PEG 4000 as precipitant. The diffraction pattern of the crystal extends to 2.30 A resolution at 100 K using synchrotron radiation at the Photon Factory. The crystals are monoclinic and belong to space group P2(1), with unit-cell parameters a = 80.5, b = 70.4, c = 83.0 A, beta = 117.8 degrees. The presence of two molecules per asymmetric unit gives a crystal volume per protein weight (VM) of 2.89 A3 Da(-1) and a solvent constant of 57.4% by volume.

Cloning, sequencing, and functional analysis of the biosynthetic gene cluster of macrolactam antibiotic vicenistatin in Streptomyces halstedii.

Vicenistatin, an antitumor antibiotic isolated from Streptomyces halstedii, is a unique 20-membered macrocyclic lactam with a novel aminosugar vicenisamine. The vicenistatin biosynthetic gene cluster (vin) spanning approximately 64 kbp was cloned and sequenced. The cluster contains putative genes for the aglycon biosynthesis including four modular polyketide synthases (PKSs), glutamate mutase, acyl CoA-ligase, and AMP-ligase. Also found in the cluster are genes of NDP-hexose 4,6-dehydratase and aminotransferase for vicenisamine biosynthesis. For the functional confirmation of the cluster, a putative glycosyltransferase gene product, VinC, was heterologously expressed, and the vicenisamine transfer reaction to the aglycon was chemically proved. A unique feature of the vicenistatin PKS is that the loading module contains only an acyl carrier protein domain, in contrast to other known PKS-loading modules containing certain activation domains. Activation of the starter acyl group by separate polypeptides is postulated as well.

Active site mapping of 2-deoxy-scyllo-inosose synthase, the key starter enzyme for the biosynthesis of 2-deoxystreptamine. Mechanism-based inhibition and identification of lysine-141 as the entrapped nucleophile.

A key enzyme in the biosynthesis of clinically important aminoglycoside antibiotics including neomycin, kanamycin, gentamicin, etc. is 2-deoxy-scyllo-inosose synthase (DOIS), which catalyzes the carbocycle formation from d-glucose-6-phosphate to 2-deoxy-scyllo-inosose (DOI). To clarify its precise reaction mechanism and crucial amino acid residues in the active site, we took advantage of a mechanism-based inhibitor carbaglucose-6-phosphate (pseudo-dl-glucose, C-6-P) with anticipation of its conversion to a reactive alpha,beta-unsaturated carbonyl intermediate. It turned out that C-6-P clearly showed time- and concentration-dependent inhibition against DOIS, and the molecular mass of the resulting modified-DOIS with C-6-P was 160 mass units larger than that of native DOIS. Thus, the expected alpha,beta-unsaturated intermediate appeared to trap a specific nucleophilic group in the active site through the Michael-type 1,4-addition. The covalently modified amino acid residue was determined to be Lys-141 by means of enzymatic digestion and subsequent LC/MS and LC/MS/MS of the digest. Also discussed are the role of Lys-141 in the substrate recognition and the reaction pathway and comparison with evolutionary related dehydroquinate synthase.

A new approach for the investigation of isoprenoid biosynthesis featuring pathway switching, deuterium hyperlabeling, and 1H NMR spectroscopy. The reaction mechanism of a novel streptomyces diterpene cyclase.

Recent methodology for the investigation of isoprenoid biosynthesis featuring pathway switching and hyperdeuteration has shown significant advantages in elucidating the reaction mechanism of a novel Streptomyces diterpene cyclase with use of precise atom-level analysis. Insight into the cyclization mechanism involved in the conversion of geranylgeranyl diphosphate (GGPP) into a clerodane hydrocarbon terpentetriene was obtained by heterologous expression in doubly engineered Streptomyces lividans of a diterpene cyclase gene derived from Streptomyces griseolosporeus, a producer of an unique diterpenoid cytotoxic antibiotic terpentecin, and by in vivo labeling with mevalonate-d(9). The cyclization involved electrophilic protonation, cationic ring closure, Wagner-Meerwein-type rearrangements, and deprotonation. A key feature was that the labeled metabolite as a mixture of predominantly deuterated mosaic molecules provided sufficient information that close analysis of the labeling pattern for each individual isoprene unit was achieved primarily by (1)H NMR spectroscopy. The cyclization of GGPP into the clerodane skeleton catalyzed by the cyclase appears to involve Si-face specific protonation, intermediates with A/B chair-boat conformation, and specific methyl and hydride migrations to give an intermediary C-4 carbocation. Subsequent collapse of the cation through specific removal of the initiating proton and final elimination of diphosphate gives rise to the terpentetriene hydrocarbon.

Identification of L-glutamine: 2-deoxy-scyllo-inosose aminotransferase required for the biosynthesis of butirosin in Bacillus circulans.

Using inverse PCR, two new genes (btrN and btrS) were identified upstream of the putative glycosyltransferase gene btrM in the butirosin-biosynthetic btr gene cluster of Bacillus circulans. The upstream gene btrS showed significant homology with stsC of Streptomyces griseus, which encodes L-glutamine:scyllo-inosose aminotransferase in the biosynthesis of streptomycin. The function of BtrS was further confirmed by heterologous expression in Escherichia coli and chemical identification of the conversion of 2-deoxy-scyllo-inosose into 2-deoxy-scyllo-inosamine. The identification of BtrS as L-glutamine:2-deoxy-scyllo-inosose aminotransferase is the first report of the aminotransferase gene responsible for 2-deoxystreptamine biosynthesis.

Significance of the 20-kDa subunit of heterodimeric 2-deoxy-scyllo-inosose synthase for the biosynthesis of butirosin antibiotics in Bacillus circulans.

A gene (btrC2) encoding the 20-kDa subunit of 2-deoxy-scyllo-inosose (DOI) synthase, a key enzyme in the biosynthesis of 2-deoxystreptamine, was identified from the butirosin-producer Bacillus circulans by reverse genetics. The deduced amino acid sequence of BtrC2 closely resembled that of YaaE of B. subtilis, but the function of the latter has not been known to date. Instead, BtrC2 appeared to show sequence similarity to a certain extent with HisH of B. subtilis, an amidotransferase subunit of imidazole glycerol phosphate synthase. Disruption of btrC2 reduced the growth rate compared with the wild type, and simultaneously antibiotic producing activity was lost. Addition of NH4Cl to the medium complemented only the growth rate of the disruptant, and both the growth rate and antibiotic production were restored by addition of yeast extract. In addition, a heterologous co-expression system of btrC2 with btrC was constructed in Escherichia coli. The simultaneously over-expressed BtrC2 and BtrC constituted a heterodimer, the biochemical features of which resembled those of DOI synthase from B. circulans more than those of the recombinant homodimeric BtrC. Despite the similarity of BtrC2 to HisH the heterodimer showed neither aminotransfer nor amidotransfer activity for 2-deoxy-scyllo-inosose as a substrate. All the observations suggest that BtrC2 is involved not only in the secondary metabolism, but also in the primary metabolism in B. circulans. The function of BtrC2 in the butirosin biosynthesis appears to be indirect, and may be involved in stabilization of DOI synthase and in regulation of its enzyme activity.

Stereostructure of a novel cytotoxic 18-membered macrolactone antibiotic FD-891.

The absolute stereochemistry of FD-891, a novel cytotoxic 18-membered macrolactone antibiotic, was determined by a synthetic approach as well as X-ray diffraction of degradative derivatives. The absolute configuration of FD-891 turned out to be as shown above. The stable conformer of FD-891 was also discussed with respect to biological activity by comparison with the structurally related concanamycin A on the bases of molecular mechanics calculations. [structure: see text]