Enzymes
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- Name help_outline D-glucose 6-phosphate Identifier CHEBI:61548 Charge -2 Formula C6H11O9P InChIKeyhelp_outline NBSCHQHZLSJFNQ-GASJEMHNSA-L SMILEShelp_outline OC1O[C@H](COP([O-])([O-])=O)[C@@H](O)[C@H](O)[C@H]1O 2D coordinates Mol file for the small molecule Search links Involved in 32 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
- Name help_outline 2-deoxy-L-scyllo-inosose Identifier CHEBI:64796 Charge 0 Formula C6H10O5 InChIKeyhelp_outline GZYCZKBRQBKGJW-FSZQNWAESA-N SMILEShelp_outline O[C@@H]1CC(=O)[C@@H](O)[C@H](O)[C@H]1O 2D coordinates Mol file for the small molecule Search links Involved in 2 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
- Name help_outline phosphate Identifier CHEBI:43474 Charge -2 Formula HO4P InChIKeyhelp_outline NBIIXXVUZAFLBC-UHFFFAOYSA-L SMILEShelp_outline OP([O-])([O-])=O 2D coordinates Mol file for the small molecule Search links Involved in 992 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
Cross-references
| RHEA:33071 | RHEA:33072 | RHEA:33073 | RHEA:33074 | |
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| Reaction direction help_outline | undefined | left-to-right | right-to-left | bidirectional |
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Publications
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Stereospecificity of hydride transfer in NAD+-catalyzed 2-deoxy-scyllo-inosose synthase, the key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics.
Huang Z., Kakinuma K., Eguchi T.
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 fi ... >> More
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. << Less
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Significance of the 20-kDa subunit of heterodimeric 2-deoxy-scyllo-inosose synthase for the biosynthesis of butirosin antibiotics in Bacillus circulans.
Tamegai H., Nango E., Koike-Takeshita A., Kudo F., Kakinuma K.
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 ... >> More
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. << Less
Biosci. Biotechnol. Biochem. 66:1538-1545(2002) [PubMed] [EuropePMC]
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Kinetic isotope effect and reaction mechanism of 2-deoxy-scyllo-inosose synthase derived from butirosin-producing Bacillus circulans.
Kudo F., Yamauchi N., Suzuki R., Kakinuma K.
The mechanism of 2-deoxy-scyllo-inosose synthase reaction, a carbocycle formation step from D-glucose-6-phosphate in the biosynthesis of the 2-deoxystreptamine aglycon of clinically important aminocyclitol antibiotics, was investigated with a partially purified enzyme from butirosin-producing Baci ... >> More
The mechanism of 2-deoxy-scyllo-inosose synthase reaction, a carbocycle formation step from D-glucose-6-phosphate in the biosynthesis of the 2-deoxystreptamine aglycon of clinically important aminocyclitol antibiotics, was investigated with a partially purified enzyme from butirosin-producing Bacillus circulans SANK 72073, Nonlabeled and double-labeled D-[4-2H, 3-15O]glucose-6-phosphate were used for cross-over experiment, and the oxime-TMS ether derivative of the 2-deoxy-scyllo-inosose product was analyzed by GC-MS. The deuterium label at C-4 of the substrate appeared to be retained at C-6 of the inosose product without scrambling of the double-labeled isotopes. Since the transient reduction of NAD+ cofactor was proved to be essential in the 2-deoxy-scyllo-inosose reaction, the hydride abstraction and returning appeared to take place within the same glucose molecule. The observed kinetic isotope effect was estimated to be kH/kD = 2.4. These results strongly suggest that this carbocycle formation is catalyzed by a single 2-deoxy-scyllo-inosose synthase enzyme with catalytic requirement of NAD+, the mechanism of which appears to be resembled closely to the 2-deoxy-scyllo-inosose synthase in the Streptomyces fradiae. << Less
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Roles of a 20 kDa protein associated with a carbocycle-forming enzyme involved in aminoglycoside biosynthesis in primary and secondary metabolism.
Tamegai H., Sawada H., Nango E., Aoki R., Hirakawa H., Iino T., Eguchi T.
2-Deoxy-scyllo-inosose (DOI) synthase participates in the biosynthesis of 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotics. The enzyme is expected to be of industrial use, because it converts a sustainable resource (glucose 6-phosphate) into carbocycle (DOI), which easily aromatizes ... >> More
2-Deoxy-scyllo-inosose (DOI) synthase participates in the biosynthesis of 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotics. The enzyme is expected to be of industrial use, because it converts a sustainable resource (glucose 6-phosphate) into carbocycle (DOI), which easily aromatizes to yield catechol. In the present study, we clarified the physiological role of a non-catalytic 20 kDa protein, BtrC2, associated with DOI synthase from the butirosin-producer Bacillus circulans. Based on the results of complementation analysis using btrC2 disruptant and western analysis against catalytic 40 kDa protein (BtrC) and BtrC2, it is suggested that BtrC2 has two functions. It is involved in the vitamin B(6) biosynthesis in primary metabolism, like homologous protein (Pdx2) in Bacillus subtilis. Additionally, it takes part in butirosin biosynthesis as stabilizer of DOI synthase by forming a heterodimer with BtrC, and qualifies B. circulans for stable and constant production of butirosin for long periods. << Less
Biosci Biotechnol Biochem 74:1215-1219(2010) [PubMed] [EuropePMC]
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Purification and characterization of 2-deoxy-scyllo-inosose synthase derived from Bacillus circulans. A crucial carbocyclization enzyme in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics.
Kudo F., Hosomi Y., Tamegai H., Kakinuma K.
The biosynthesis of 2-deoxystreptamine, the central aglycon of a major group of clinically important aminoglycoside antibiotics, commences with the initial carbocycle formation step from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose. This crucial step is known to be catalyzed by 2-deoxy-scyllo-i ... >> More
The biosynthesis of 2-deoxystreptamine, the central aglycon of a major group of clinically important aminoglycoside antibiotics, commences with the initial carbocycle formation step from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose. This crucial step is known to be catalyzed by 2-deoxy-scyllo-inosose synthase, which has not yet been characterized so far. Reported in this paper is the first purification of 2-deoxy-scyllo-inosose synthase from butirosin-producing Bacillus circulans SANK 72073 to electrophoretic homogeneity. The enzyme was isolated as a heterodimeric protein comprising from a 23 kDa- and a 42 kDa polypeptide chains. The Km of the enzyme for D-glucose-6-phosphate was estimated to be 9.0 x 10(-4) M and that for NAD+ 1.7 x 10(-4) M, kcat for D-glucose-6-phosphate being 7.3 x 10(-2) s(-1). The presence of Co2+ was essential for the enzyme activity, but Zn2+ was totally inhibitory. While the reaction mechanisms are quite similar, 2-deoxy-scyllo-inosose synthase appears to be distinct from dehydroquinate synthase in the shikimate pathway, with respect to the quaternary structure, metal ion requirement, and the kinetic parameters. << Less
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Expression of 2-deoxy-scyllo-inosose synthase (kanA) from kanamycin gene cluster in Streptomyces lividans.
Thuy M.L., Kharel M.K., Lamichhane R., Lee H.C., Suh J.W., Liou K., Sohng J.K.
An actinomycetes expression vector (pIBR25) was constructed and applied to express a gene from the kanamycin biosynthetic gene cluster encoding 2-deoxy-scyllo-inosose synthase (kanA) in Streptomyces lividans TK24. The expression of kanA in pIBR25 transformants reached a maximum after 72 h of cultu ... >> More
An actinomycetes expression vector (pIBR25) was constructed and applied to express a gene from the kanamycin biosynthetic gene cluster encoding 2-deoxy-scyllo-inosose synthase (kanA) in Streptomyces lividans TK24. The expression of kanA in pIBR25 transformants reached a maximum after 72 h of culture. The plasmid pIBR25 showed better expression than pSET152, and resulted in the formation of insoluble KanA when it was expressed in Escherichia coli. This strategy thus provides a valuable tool for expressing aminoglycoside-aminocyclitols (AmAcs) biosynthetic genes in Streptomyces spp. << Less
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Molecular cloning of the gene for the key carbocycle-forming enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics and its comparison with dehydroquinate synthase.
Kudo F., Tamegai H., Fujiwara T., Tagami U., Hirayama K., Kakinuma K.
The 2-deoxystreptamine aglycon is a common structural feature found in aminocyclitol antibiotics including neomycin, kanamycin, tobramycin, gentamicin, sisomicin, butirosin and ribostamycin. A key enzyme involved in the biosynthesis of the 2-deoxystreptamine moiety is 2-deoxy-scyllo-inosose (DOI) ... >> More
The 2-deoxystreptamine aglycon is a common structural feature found in aminocyclitol antibiotics including neomycin, kanamycin, tobramycin, gentamicin, sisomicin, butirosin and ribostamycin. A key enzyme involved in the biosynthesis of the 2-deoxystreptamine moiety is 2-deoxy-scyllo-inosose (DOI) synthase which catalyses the carbocycle formation from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose. The recent success of isolating the 2-deoxy-scyllo-inosose synthase from Bacillus circulans prompted us to clone the gene responsible for this important enzyme by the use of reverse genetics approach. With the aid of DNA probes constructed on the basis of the amino-terminal sequence of the purified 42 kDa subunit of the enzyme, the responsible gene btrC was successfully cloned. Subsequently the btrC gene was heterologously expressed in Escherichia coli, and the 2-deoxy-scyllo-inosose synthase activity of the recombinant polypeptide was confirmed by chemical analysis. The btrC gene encodes a protein composed of 368 amino acids with a molecular mass of 40.7 kDa. Our previous proposal for the similarity of 2-deoxy-scyllo-inosose synthase to dehydroquinate synthase has been confirmed on the basis of their amino acid sequences. Significant differences in the sequences can also be observed however, particularly in the crucial substrate recognition regions. Comparison of the BtrC sequence with those of biosynthetic enzymes for other related microbial products is also discussed. << Less
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Structure of 2-deoxy-scyllo-inosose synthase, a key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics, in complex with a mechanism-based inhibitor and NAD+.
Nango E., Kumasaka T., Hirayama T., Tanaka N., Eguchi T.
A key enzyme in the biosynthesis of clinically important aminoglycoside antibiotics is 2-deoxy-scyllo-inosose synthase (DOIS), which catalyzes carbocycle formation from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose through a multistep reaction. This reaction mechanism is similar to the catalysis ... >> More
A key enzyme in the biosynthesis of clinically important aminoglycoside antibiotics is 2-deoxy-scyllo-inosose synthase (DOIS), which catalyzes carbocycle formation from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose through a multistep reaction. This reaction mechanism is similar to the catalysis by dehydroquinate synthase (DHQS) of the cyclization of 3-deoxy-D-arabino-heputulosonate-7-phosphate to dehydroquinate in the shikimate pathway, but significant dissimilarity between these enzymes is also known, particularly in the stereochemistry of the phosphate elimination reaction and the cyclization. Here, the crystal structures of DOIS from Bacillus circulans and its complex with the substrate analog inhibitor carbaglucose-6-phosphate, NAD+, and Co2+ have been determined to provide structural insights into the reaction mechanism. The complex structure shows that an active site exists between the N-terminal and C-terminal domains and that the inhibitor coordinates a cobalt ion in this site. Two subunits exist as a dimer in the asymmetric unit. The two active sites of the dimer were observed to be different. One contains a dephosphorylated compound derived from the inhibitor and the other includes the inhibitor without change. The present study suggested that phosphate elimination proceeds through syn-elimination assisted by Glu 243 and the aldol condensation proceeds via a boat conformation. Also discussed are significant similarities and dissimilarities between DOIS and DHQS, particularly in terms of the structure at the active site and the reaction mechanism. << Less