The fifth gene of the iol operon of Bacillus subtilis, iolE, encodes 2-keto-myo-inositol dehydratase
Ken-ichi Yoshida1,
Masanori Yamaguchi2,
Hideki Ikeda1,
Kaoru Omae1,
Ken-ichi Tsurusaki3 and
Yasutaro Fujita1
1 Department of Biotechnology, Faculty of Life Science and Biotechnology, Fukuyama University, 985 Sanzo, Higashimura-cho, Fukuyama-shi, Hiroshima 729-0292, Japan
2 Central Research Laboratories, Hokko Chemical Industry Co., Ltd, 2165 Toda, Atsugi-shi, Kanagawa 243-0023, Japan
3 Department of Environment and Information Science, Faculty of Human Culture and Sciences, Fukuyama University, 985 Sanzo, Higashimura-cho, Fukuyama-shi, Hiroshima 729-0292, Japan
Correspondence
Ken-ichi Yoshida
kyoshida{at}bt.fubt.fukuyama-u.ac.jp
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ABSTRACT
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The myo-inositol catabolism pathway of Bacillus subtilis has not been fully characterized but was proposed to involve step-wise multiple reactions that finally yielded acetyl-CoA and dihydroxyacetone phosphate. It is known that the iolABCDEFGHIJ operon is responsible for the catabolism of inositol. IolG catalyses the first step of myo-inositol catabolism, the dehydrogenation of myo-inositol, producing 2-keto-myo-inositol (inosose). The second step was thought to be the dehydration of inosose. Genetic and biochemical analyses of the iol genes led to the identification of iolE, encoding the enzyme for the second step of inositol catabolism, inosose dehydratase. The reaction product of inosose dehydratase was identified as D-2,3-diketo-4-deoxy-epi-inositol.
Abbreviations: ESI-TOF, electrospray ionization-time-of-flight
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INTRODUCTION
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myo-Inositol is abundant in soil and also common and essential in plants. Several micro-organisms, including Bacillus subtilis (Yoshida et al., 1997
), Cryptococcus melibiosum (Vidal-Leiria & van Uden, 1973
), Aerobacter aerogenes (reclassified as Enterobacter aerogenes/Klebsiella mobilis) (Berman & Magasanik, 1966a
), Rhizobium leguminosarum bv. viciae (Poole et al., 1994
), Sinorhizobium meliloti (Galbraith et al., 1998
) and Sinorhizobium fredii (Jiang et al., 2001
), can grow on inositol as the carbon source. It was thought that bacterial inositol catabolism is only required for efficient utilization of this compound. However, the inositol dehydrogenase of S. fredii not only catalyses the initial reaction step of inositol catabolism but also is involved in nitrogen fixation and competitiveness to nodulate soybeans (Jiang et al., 2001
). Furthermore, MocA and MocC of S. meliloti which participate in degradation of rhizopine (L-3-O-methyl-scyllo-inosamine), a symbiosis-specific compound found in alfalfa nodules, exhibited significant similarities to IolG and IolE involved in inositol catabolism of B. subtilis, respectively (Rossbach et al., 1994
; Yoshida et al., 1997
), and the inositol catabolism pathway was tightly linked with rhizopine utilization in S. meliloti (Galbraith et al., 1998
). These facts implied an interesting relationship between bacterial inositol catabolism and plantbacteria symbiosis for nitrogen fixation.
Inositol catabolism in A. aerogenes was studied biochemically, and a pathway of the catabolism finally yielding acetyl-CoA and dihydroxyacetone phosphate has been proposed as illustrated in Fig. 1
(Anderson & Magasanik, 1971a
, b
; Berman & Magasanik, 1966a
, b
). However, our knowledge of the molecular genetics of bacterial inositol catabolism has been restricted to B. subtilis (Fujita et al., 1991
; Yoshida et al., 1997
, 1999
, 2002
). In B. subtilis, the iol divergon, comprising the operons iolABCDEFGHIJ and iolRS (Fig. 2a
) (Yoshida et al., 1997
), and the iolT gene (Yoshida et al., 2002
) were shown to be involved in inositol catabolism. A repressor encoded by iolR was responsible for transcriptional regulation of both the iol divergon and iolT (Yoshida et al., 1999
, 2002
). In the absence of inositol in the growth medium, the IolR repressor bound to the operator site within the promoter regions to repress the transcription, while in its presence inositol was taken into the cell and converted to a catabolic intermediate that acted as an inducer antagonizing IolR for induction of the iol divergon and iolT (Yoshida et al., 1999
, 2002
). Thus, inactivation of iolR made the iol transcriptions constitutive (Yoshida et al., 1997
).

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Fig. 1. myo-Inositol catabolism pathway and function of the B. subtilis iol genes. The pathway of inositol catabolism in A. aerogenes based on the previous proposal of Anderson & Magasanik (1971b) is shown with the structures of the intermediates [compounds (1) to (8)]. Assuming that the pathway in B. subtilis is similar to that in A. aerogenes, B. subtilis iol genes proven and deduced (underlined and parenthesized, respectively) to encode the enzymes involved in some of the reaction steps are shown (Yoshida et al., 1997 , 2002 ). Compound: (1), myo-inositol (carbon numbering is indicated); (2), inosose; (3), D-2,3-diketo-4-deoxy-epi-inositol; (4), 2-deoxy-5-keto-D-gluconic acid; (5), 2-deoxy-5-keto-D-gluconic acid 6-phosphate; (6), dihydroxyacetone phosphate; (7), malonic semialdehyde; (8), acetyl-CoA.
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Fig. 2. Gene organization of the iol region and -galactosidase synthesis in strains of B. subtilis. (a) Gene organization of the iol region of B. subtilis 60015. Gene organization and transcription of the iol divergon, the iol and iolRS operons, are shown schematically. Piol and PiolRS denote the iol and iolRS promoters, respectively. (b) Chromosomal arrangement of the iol region of a pMUTIN-integrant FU429. The iolB gene was split into iolB' and 'iolB and disrupted after Campbell-type integration of a pMUTIN2 derivative. Instead of iolB, lacZ is expressed under the control of Piol. The spac promoter (Pspac), regulated by LacI and thus induced in the presence of IPTG, is responsible for the expression of the other iol genes downstream of iolB. (ori) and (amp) are not functioning in B. subtilis. (c) -Galactosidase activity in protein extracts of the cells of strains 60015 and FU428 to FU437 (Table 1 , each iol gene disrupted by pMUTIN integration is shown beneath in parentheses), grown in S6 medium containing 0·5 % Casamino acids supplemented with 10 mM inositol (Iol), 1 mM IPTG (IPTG) or 10 mM inositol plus 1 mM IPTG (Iol+IPTG), or no additional compounds (None), was measured. Mean values ±SD of three independent measurements are shown.
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The enzymes involved in inositol catabolism in B. subtilis have not been completely elucidated. Inositol dehydrogenase encoded by iolG was responsible for the first step of the degradation cascade [conversion of compound (1) to (2) in Fig. 1
] (Ramaley et al., 1979
; Fujita et al., 1991
; Yoshida et al., 1997
), and iolT and iolF encoded the primary and secondary inositol transporters, respectively (Fig. 1
) (Yoshida et al., 2002
). Based on an assumption that the catabolism proceeded similarly as in A. aerogenes (Berman & Magasanik, 1966a
) and the results of the homology search for the iol genes (Yoshida et al., 1999
), iolC, iolJ and iolA were deduced to be involved in the fourth, fifth and sixth steps, respectively (Fig. 1
). The second step was thought to be the dehydration of inosose catalysed by inosose dehydratase [conversion of compound (2) to (3) in Fig. 1
]. Inosose dehydratase activity was demonstrated in A. aerogenes (Berman & Magasanik, 1966a
), S. meliloti (Galbraith et al., 1998
) and R. leguminosarum bv. viciae (Poole et al., 1994
). However, no such gene encoding the enzyme in these bacteria has been identified so far. In this study, we found that B. subtilis iolE encoded inosose dehydratase, the reaction product of which was identified as D-2,3-diketo-4-deoxy-epi-inositol [compound (3) in Fig. 1
].
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METHODS
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Bacterial strains, plasmids and growth media.
Strains and plasmids used in this work are listed in Table 1
. B. subtilis cells were grown on tryptose blood agar base (Difco) plates supplemented with 0·18 % glucose (TBABG) at 30 °C and in S6 medium (Fujita & Freese, 1981
) containing 0·5 % Casamino acids with or without 10 mM inositol at 37 °C with shaking. Escherichia coli cells were grown in LuriaBertani (LB) (Sambrook & Russell, 2001
) and TGA (Kaempfer & Magasanik, 1967
) media at 37 °C with shaking. When needed to select and grow the mutants and transformants, chloramphenicol (10 mg l-1), erythromycin (0·3 mg l-1) and ampicillin (50 mg l-1) were added to the culture media.
Construction of B. subtilis strains.
Each of the iol genes was disrupted through integration of plasmid pMUTIN as described previously (Yoshida et al., 2000
). Part of each of the iol genes was amplified by PCR with a specific primer pair and DNA of strain 168 as a template; nucleotide sequences of the primers used are available at the BSORF website (http://bacillus.genome.ad.jp/). The PCR products were trimmed with an appropriate enzyme set of HindIII and BamHI (or BglII), and ligated with the HindIIIBamHI arm of pMUTIN1 (or pMUTIN2) (Vagner et al., 1998
). Each of the recombinant plasmids was used to transform strain 168 to erythromycin-resistance to obtain pMUTIN integrants into each of iol genes. The resulting disruptant strains were IOLAd, IOLBd, IOLCd, IOLDd, IOLEd, IOLFd, IOLGd, IOLHd, IOLId and IOLJd (http://bacillus.genome.ad.jp/). DNA of these strains was then used to transform 60015 to erythromycin-resistance to get a series of the 10 iol disruptant strains (FU428 to FU437 in Table 1
).
Strain YF111 is one of the iol mutants defective in inositol utilization obtained by ethyl methanesulfonate mutagenesis and subsequent detection of colonies unable to ferment inositol on tetrazolium-containing plates as described previously (Fujita & Fujita, 1983
). This iol mutation was localized to iolE by its complementation test through transformation using various PCR fragments covering each of the iol genes, as described previously (Fujita et al., 1998
). Nucleotide sequencing revealed a missense mutation of iolE [Pro242 (CCC) to Leu (CTC)], which was designated iolE41. Strain YF111 (iolE41) was transformed with DNA of strain YF244 (iolR : : cat), resulting in strain YF256 (iolR : : cat iolE41).
Expression of each iol gene in E. coli cells.
DNA fragments covering each of the iolB, iolC, iolD and iolE coding regions associated with its ShineDalgarno sequence with flanking EcoRI and BamHI sites at the head and tail, respectively, were amplified from DNA of strain 60015 by PCR using a specific primer pair (Table 2
). Each of the fragments was trimmed with EcoRI and BamHI, and ligated with the EcoRIBamHI arm of pUC18. The ligated DNA was used for the transformation of E. coli strain JM109 to ampicillin-resistance to result in plasmids pIOLB, pIOLC, pIOLD and pIOLE, which carried the respective iol genes placed under the control of the pUC18-borne lac promoter. Correct construction of the plasmids was confirmed by nucleotide sequencing. To determine the expression of the iolB, iolC, iolD and iolE genes, E. coli JM109 cells carrying plasmid pIOLB, pIOLC, pIOLD or pIOLE were grown in TGA medium containing ampicillin with and without 1 mM IPTG. Protein extracts of the cells were prepared as described previously (Yoshida et al., 1999
) and analysed by SDS-PAGE.
Enzyme assay.
-Galactosidase activity in crude protein extracts of B. subtilis cells was determined spectrophotometrically as described previously (Yoshida et al., 2000
). For assaying of inosose dehydratase, E. coli JM109 cells carrying pUC18, pIOLB, pIOLC, pIOLD or pIOLE were grown with IPTG, and the protein extracts were prepared as described. B. subtilis cells were grown with and without inositol, and the extracts were prepared in the same way for inositol dehydrogenase assay (Yoshida et al., 1997
). The dehydratase assay was performed by the procedures modified from those of Berman & Magasanik (1966a)
. The assay mixture contained the protein extract, 50 mM Tris/HCl (pH 8·0), 0·1 mM glutathione, 0·05 mM CoCl2 and 0·5 mM inosose (3 mM for B. subtilis extracts). Inosose was prepared by the bacterial oxidation of inositol using Gluconobacter oxydans ATCC 621 as described by Posternak (1962)
. Dehydration of inosose was determined by monitoring the ultraviolet absorbance of the reaction product at 260 nm. Upon addition of inosose, the rate of increase of the absorbance was measured, and the molar concentration of the product was calculated from its estimated molar absorption coefficient of 6000 (Berman & Magasanik, 1966a
).
Isolation and identification of the IolE reaction product.
The IolE reaction product was produced and isolated as follows. In a test tube with a screw-cap, 400 mg (2·25 mmol) inosose was dissolved in 8 ml water. The solution was adjusted to pH 7·5, mixed with 2 ml of enzyme solution containing approximately 30 mg of protein extract of strain JM109(pIOLE) grown with 1 mM IPTG, saturated with nitrogen gas and incubated at 26 °C for 12 h. The reaction was terminated by the addition of 0·5 ml Duolite C20 resin (Auchtel Products). After the solution had been acidified, the tube was incubated at 55 °C for 10 min, then centrifuged to remove denatured proteins. The supernatant was successively passed through activated charcoal (2 ml), Duolite C20 (4 ml) and Duolite A368S (7 ml), which had been layered in a small column, to remove hydrophobic and ionic substances. The solution eluted from the column was concentrated and freeze-dried, to obtain the IolE reaction product as white crystals.
Reduction of the IolE reaction product was carried out as follows. One hundred milligrams (0·625 mmol) of the product was dissolved in 5 ml water, and 14·3 mg (0·375 mmol) NaBH4 was added, which was enough to reduce 1·25 mmol of carbonyl groups. The reaction mixture was stirred in an ice-bath for 10 min and then at room temperature for 10 min. The solution was acidified by the addition of 0·2 ml of 1 M HCl, and mixed with 1 ml Duolite C20 resin to remove basic substances. The solution was passed through a paper filter, to remove the resin, and mixed with 1 ml Duolite A116 resin, to remove acidic substances. The solution was filtered once again, then aliquots of it were subjected to HPLC analysis. HPLC was performed on a Wakosil 5NH2 column, 4·6x250 mm (Wako Pure Chemical Industries), kept at 40 °C with 80 % acetonitrile as solvent at a flow rate of 2 ml min-1. Refractive index and optical rotation detectors were used to monitor the elution profile. Four quercitol stereoisomers [(+)-proto-quercitol, (-)-vibo-quercitol, (+)-epi-quercitol and scyllo-quercitol (custom preparation, Hokko Chemical Industry) (Takahashi et al., 1998
)] were used as authentic samples to identify the compounds produced by the reduction. The eluted fractions corresponding to a peak at retention time 7·4 min were collected, concentrated and dried to obtain 2·5 mg of the reduced compound [(+)-proto-quercitol]. Its 300 MHz 1H-NMR and 75 MHz 13C-NMR spectra were obtained in D2O (JNM-LA300 FT NMR System; JEOL).
Phenylhydrazone of the IolE reaction product was obtained as follows. Thirty milligrams (0·19 mmol) of the product was dissolved in 1 ml water, and 0·25 ml of 2·4 M phenylhydrazine in acetic acid (0·6 mmol) was added and mixed well. After being chilled for 5 h, phenylhydrazone appearing in the form of a reddish-yellow precipitate was collected by filtration and washed with a small amount of 50 % methanol to obtain greenish-yellow needle-like crystals. Its 300 MHz 1H-NMR spectrum was obtained in CD3OD.
Mass spectra of the IolE reaction product and its phenylhydrazone were determined by ESI-TOF/MS (Mariner Biospectrometry Workstation; Applied Biosystems). The IolE reaction product was dissolved in 50 % acetonitrile containing 1 % triethylamine for anion analysis, while the phenylhydrazone was in 50 % acetonitrile containing 0·5 % formic acid for cation analysis.
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RESULTS
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IolB, IolC, IolD, IolE and IolG were involved in the initial steps of inositol catabolism
myo-Inositol and inosose, the product of the first reaction step, did not antagonize the operator-binding of IolR, indicating that neither of them functioned as the inducer (Yoshida et al., 1999
). Thus, the second step presumably catalysed by inosose dehydratase had to be involved in the inducer production. In order to find the inosose dehydratase gene, we first attempted to systematically select out the genes required for the inducer production. Each of the iol genes was disrupted through integration of plasmid pMUTIN, resulting in strains FU428 to FU437 (Table 1
). As an example, chromosomal arrangement of the iol region of strain FU429 with the disruption of iolB is shown in Fig. 2(b)
. The pMUTIN-borne lacZ reporter gene (Vagner et al., 1998
) integrated into each of the disrupted genes was used to monitor transcription from the iol promoter. In addition, the spac promoter under the control of LacI drove expression of the genes downstream of the disrupted one so that they were induced in the presence of IPTG (Vagner et al., 1998
). Therefore, only when the disrupted gene was not required for the inducer production would
-galactosidase be induced in the presence of inositol alone and/or both inositol and IPTG. The disruptants were grown in the presence and absence of inositol and/or IPTG, then
-galactosidase activity in the cell extracts was measured (Fig. 2c
). Only in the iolA and iolF disruptants were both inositol and IPTG required for induction of
-galactosidase, suggesting that neither iolA nor iolF but some of their downstream genes might be involved in inducer production. [iolF encodes one of the two inositol transporters, which was dispensable when the other one (iolT) functioned (Yoshida et al., 2002
). The relatively higher activity detected in the iolF disruptant grown with inositol alone was probably due to leaky expression of iolG encoding inositol dehydrogenase owing to read-through of transcription from the erythromycin-resistance gene, which was detected in the integrants of plasmid pMUTIN1 (Vagner et al., 1998
).] However, only inositol was enough to induce
-galactosidase in the iolH, iolI and iolJ disruptants, suggesting that none of the three genes might be required for the inducer production. On the other hand,
-galactosidase was never induced in the iolB, iolC, iolD, iolE and iolG disruptants even in the presence of both inositol and IPTG. Therefore, it was highly likely that IolB, IolC, IolD, IolE and IolG were all involved in the initial reaction steps of inositol catabolism to produce the inducer. As IolG is inositol dehydrogenase which catalyses the first reaction step (Fujita et al., 1991
; Yoshida et al., 1997
), one of IolB, IolC, IolD and IolE could be inosose dehydratase catalysing the second step.
iolE encoded inosose dehydratase
Each of the iolB, iolC, iolD and iolE coding regions together with its ShineDalgarno sequence was cloned into the multi-cloning site of pUC18 to provide pIOLB, pIOLC, pIOLD and pIOLE, respectively. Thus, the cloned genes were placed under the control of the lac promoter in E. coli. The production of the iol gene products in E. coli cells was confirmed by the appearance of extra protein bands on SDS-PAGE corresponding to the expected sizes from their respective amino acid sequences (Fig. 3
). We found, unexpectedly, that the open reading frame (ORF) of iolD was probably 171 bp longer toward upstream than that reported (Kunst et al., 1997
) due to misassignment of its translation start site, because expression of the reported ORF of iolD in E. coli cells produced no extra protein detected upon SDS-PAGE (data not shown). However, when we cloned a DNA fragment into pUC18, which contained not only the previously reported iolD gene but also the 171 bp located upstream of it as well as an almost-perfect ribosome-binding site (AAAGGTGG) located a further 8 bp upstream of the newly presumed GTG start codon, we observed an additional protein band in crude extracts of the E. coli transformant exhibiting the size (70 kDa) expected for the protein encoded by the 5'-extended iolD gene (Fig. 3
, lane 10).

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Fig. 3. Production of the iolB, iolC, iolD and iolE gene products in E. coli. Cells of E. coli JM109 carrying pUC18 (lanes 1, 2, 7 and 8), pIOLB (lanes 3 and 4), pIOLC (lanes 5 and 6), pIOLD (lanes 9 and 10) or pIOLE (lanes 11 and 12) were grown in the presence (lanes 2, 4, 6, 8, 10 and 12) and absence (lanes 1, 3, 5, 7, 9 and 11) of 1 mM IPTG. Protein extracts from the cells were prepared and analysed by SDS-PAGE; each of the lanes contained 50 µg of the protein extract. Lane M, size marker proteins. Protein bands of IolB, IolC, IolD and IolE are indicated with arrows on the right-hand side of both panels.
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Inosose dehydratase activity in the protein extracts of the E. coli cells expressing one of iolB, iolC, iolD and iolE was measured (Table 3
). The results clearly indicated that only the extract prepared from cells expressing iolE contained a large amount of inosose dehydratase. On the other hand, the enzyme activity in the cells of B. subtilis 60015 (wild-type) was induced when the cells were grown with inositol (Table 3
). The activity in strain YF244 (iolR : : cat) was high and constitutive, whereas that in a mutant, YF256 (iolR : : cat iolE41), turned out to be negligible in spite of the constitutive iol transcription caused by the iolR inactivation (Yoshida et al., 1999
). From these results, we concluded that the iolE gene encoded B. subtilis inosose dehydratase, which was induced in the presence of inositol in the growth medium.
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Table 3. Inosose dehydratase activity in E. coli cells expressing the B. subtilis iol genes and in strains of B. subtilis
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Properties of B. subtilis inosose dehydratase produced in E. coli cells
The inosose dehydratase activity in the crude extract of E. coli cells expressing iolE was remarkably high, while those in the other extracts were negligible (Table 3
). Thus, we attempted to determine the basic characteristics of B. subtilis inosose dehydratase produced in E. coli cells without its purification. The assay was conducted under the conditions analogous to those established in an earlier study (Berman & Magasanik, 1966a
). The activity peak was seen at pH 7·58 (Fig. 4
). A LineweaverBurk plot for the enzyme gave an apparent Km value for inosose of 1·65±0·27 mM at pH 8 (data not shown). It was reported that A. aerogenes inosose dehydratase required 0·1 mM glutathione and 0·05 mM Co2+ or Mn2+ ions for maximal activity (Berman & Magasanik, 1966a
). The enzyme activity in the protein extract was about twice higher in the presence of 0·1 mM glutathione plus 0·05 mM CoCl2 or MnCl2 than in their absence (data not shown). MnCl2 appeared to be less suitable to this enzyme reaction than CoCl2 (data not shown). In addition, EDTA was able to counteract these effects of the metal ions partially (data not shown).

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Fig. 4. pH dependency of B. subtilis inosose dehydratase. The protein extract prepared from E. coli JM109 expressing B. subtilis iolE was used for the inosose dehydratase assay at various pH values with 0·5 mM inosose. The relative activities (percentages to the activity at pH 8) determined in 50 mM potassium/HEPES and 50 mM Tris/HCl buffers are shown with open and solid bars, respectively. Because the reaction product decayed rapidly at higher pH values, it was hard to determine the optimum pH accurately. Representative results from three independent experiments are shown.
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Identification of the product of the IolE inosose dehydratase reaction
The enzymic conversion of inosose by the extract of E. coli expressing iolE was so efficient that inosose was completely consumed after 12 h of the reaction under the conditions described above. The conversion of 400 mg (2·25 mmol) of inosose allowed us to isolate 245 mg (1·53 mmol) of the product. The IolE reaction product was expected to be D-2,3-diketo-4-deoxy-epi-inositol (C6H8O5). The ESI-TOF mass spectrum of the isolated product gave a peak of an anion at m/z 159·0297. This anion was probably the molecular ion of D-2,3-diketo-4-deoxy-epi-inositol ([C6H8O5-H]-, calculated mass m/z 159·0288). Then, the product was reduced with NaBH4 and subjected to HPLC analysis as described above. Since the product was expected to possess two carbonyl groups, the reduction could give four stereoisomers of quercitol. The HPLC elution profile gave four peaks at retention times of 7·4, 8·6, 9·0 and 9·3 min exhibiting optical rotation in 80 % acetonitrile
+21·9±1·9°, -61·1±2·3°, +6·0±0·6° and +0·6±0·7° (mean±SD of seven independent experiments), respectively, while the authentic samples of four stereoisomers of quercitol, (+)-proto-quercitol, (-)-vibo-quercitol, (+)-epi-quercitol and scyllo-quercitol [compounds (9), (10), (11) and (12), respectively, in Fig. 5
], gave peaks at 7·4, 8·6, 9·0 and 9·3 min exhibiting
+23·8±0·9°, -64·5±0·8°, +4·7±0·8° and +0·2±1·0°, respectively. Furthermore, we isolated the compound contained in the first peak at retention time of 7·4 min, and its 1H-NMR and 13C-NMR spectra (data not shown) coincided with those reported for (+)-proto-quercitol (McCasland et al., 1968
). These results strongly suggested that the IolE reaction product could be reduced to become the four stereoisomers of quercitol, implying that the reduced two carbonyl groups could have been located at the positions corresponding to C-2 and C-3 of the quercitols (Fig. 5
) [carbon numbering was defined according to that of myo-inositol, compound (1) in Fig. 1
].

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Fig. 5. Four stereoisomers of quercitol produced after the reduction of the IolE reaction product. Compounds: (9), (+)-proto-quercitol [carbon numbering was defined according to that of myo-inositol, compound (1) in Fig. 1 ]; (10), (-)-vibo-quercitol; (11), (+)-epi-quercitol; (12), scyllo-quercitol. Hydroxyl groups judged to be produced by the reduction of the two carbonyl groups are indicated with asterisks.
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Since the 1H-NMR spectrum of the IolE reaction product itself (data not shown) was too complicated to identify the product, probably due to ketoenol tautomerization, the product was converted to its phenylhydrazone for determination of its structure. Starting from 30 mg (0·19 mmol) of the product, we obtained 29 mg (0·086 mmol) of its phenylhydrazone. The ESI-TOF mass spectrum of the phenylhydrazone gave a peak of a cation at m/z 341·1615. This cation corresponded to the molecular ion of a bisphenylhydrazone ([C18H20N4O3+H]+, calculated mass m/z 341·1608), confirming the two carbonyl groups. The 1H-NMR spectrum of the bisphenylhydrazone revealed five protons contained in it, and the coupling patterns of the two primary protons (H-4 and H-5 in Fig. 6
) suggested that both of them could belong to a -CH2- group vicinal to one of the carbonyl groups. Thus, the two carbonyl groups were assigned to C-2 and C-3, and the -CH2- group was assigned to C-4 (Fig. 6
), referring to the structures of inosose [2-keto-myo-inositol (1,3,5/4,6-pentahydroxycyclohexan-2-one), compound (2) in Fig. 1
] and the four stereoisomers of quercitol produced by the reduction of the IolE reaction product (Fig. 5
). Starting from the -CH2- protons, the other three protons (H-3, H-2 and H-1) were assigned one by one to the positions according to their chemical shift and coupling patterns (Fig. 6
). The assignment was also confirmed by the appearance of cross peaks in a COSY spectrum of the bisphenylhydrazone (data not shown). Determination of the coupling constants among the protons established the configuration of the bisphenylhydrazone (Fig. 6
). Finally, the structure of the bisphenylhydrazone was defined as shown in Fig. 6
. Taken together, all the results indicated that the IolE reaction product was D-2,3-diketo-4-deoxy-epi-inositol [compound (3) in Fig. 1
], which was identical to the product of the A. aerogenes inosose dehydratase reaction (Berman & Magasanik, 1966a
).

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Fig. 6. Structure determination of the phenylhydrazone of the IolE reaction product. A summarized table for the 1H-NMR spectrum analysis of the phenylhydrazone of the IolE reaction product and its defined structural formula are given. Carbon numbering was defined according to that of myo-inositol [compound (1) in Fig. 1 ].
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DISCUSSION
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We identified that the B. subtilis iolE gene encoded inosose dehydratase (Table 3
) for the second reaction step of the inositol catabolism pathway (Fig. 1
). The reaction product was identified, by determination of the structure of its reduced forms (Fig. 5
) and its bisphenylhydrazone (Fig. 6
), to be D-2,3-diketo-4-deoxy-epi-inositol [compound (3) in Fig. 1
], which was identical to that reported for the A. aerogenes enzyme (Berman & Magasanik, 1966a
). The protein extract of E. coli cells producing the B. subtilis inosose dehydratase (Fig. 3
) was used to determine some properties of the enzyme. The apparent pH optimum for this enzyme was pH 7·58 (Fig. 4
), and its Km value for inosose was 1·65±0·27 mM at pH 8, while the A. aerogenes one gave a pH optimum at pH 66·2 and a Km value of 0·15 mM at pH 6 (Berman & Magasanik, 1966a
). These differences in the pH optima and Km values for inosose might possibly reflect those in the actual environmental conditions of the two micro-organisms. However, the B. subtilis enzyme required 0·1 mM glutathione and 0·05 mM Co2+ or Mn2+ for maximal activity, and the cofactor requirement was almost the same as reported for the A. aerogenes enzyme. We did not examine substrate specificities for the B. subtilis enzyme, because other substrates such as stereoisomers of inosose, which could be utilizable for our assay system depending on the ultraviolet absorbance of the product, were not available.
A protein similarity search in the SWISSPROT database (Release 40.0+/02-10) using the FASTA program (Pearson & Lipman, 1988
) revealed that B. subtilis IolE exhibited the highest optimized score of 677 to S. meliloti MocC (Rossbach et al., 1994
). The amino acid residue Pro242 of IolE (substituted to Leu in the iolE41 mutation) was indispensable for the enzyme activity. In the amino acid sequence of MocC, not only the residue corresponding to Pro242 but also its flanking residues were well conserved. MocC is responsible for the utilization of rhizopine (Rossbach et al., 1994
), which might function as a dehydratase for an inosose-like intermediate formed in rhizopine catabolism in S. meliloti. Furthermore, MocA, another enzyme needed for rhizopine utilization, shares a high similarity with IolG, inositol dehydrogenase (Rossbach et al., 1994
). Hence, it is assumable that the substrate of MocC might be the MocA reaction product. These facts support well the previous idea of Galbraith et al. (1998)
that the inositol catabolism pathway was tightly linked with rhizopine utilization in S. meliloti. IolE was found to possess a sequence motif for the inner-membrane component signature of the binding-protein-dependent transport systems (PROSITE: PS00402) from the 33rd to 61st residues. However, this motif was not conserved in MocC, so its significance remained to be examined. On the other hand, the paralogue of IolE exhibiting the highest FASTA optimized score of 118 was IolH. IolH was unlikely to be another inosose dehydratase, because the iolE41 mutant did not possess this enzyme activity and could not grow on inositol (data not shown). We do not know the function of IolH, but it might be an enzyme which catalyses a reaction involving a compound related to inosose.
The B. subtilis iolB, iolC, iolD, iolE and iolG genes were all found to be indispensable for the production of the inducer (Fig. 2
). Also, the first and second reaction steps in the inositol catabolism pathway were proven to be catalysed by inositol dehydrogenase and inosose dehydratase encoded by iolG and iolE, respectively (Fig. 1
). Thus, the remaining three genes, iolB, iolC and iolD, could be responsible for the later steps leading to the production of the inducer. In A. aerogenes, the product of the inosose dehydratase reaction was successively hydrolysed by a hydrolase, phosphorylated by a kinase, then cleaved by an aldolase (Fig. 1
) (Anderson & Magasanik, 1971a
, b
; Berman & Magasanik, 1966b
). B. subtilis IolC exhibited high similarity to fructokinases (Yoshida et al., 1997
). IolJ, exhibiting significant similarity to aldolases (Yoshida et al., 1997
), was not required for inducer production (Fig. 2
). In addition, B. subtilis IolR, the repressor of the iol operon, belongs to the DeoR family of repressors. Members of this family are known to interact with an inducer of phosphorylated sugar (van Rooijen & de Vos, 1990
). Therefore, it is likely that IolB, IolC and IolD might be involved in the successive hydrolysis and phosphorylation steps (Fig. 1
). If the inositol catabolism of B. subtilis was taking place similar to that in A. aerogenes, the phosphorylated intermediate acting as the inducer interacting with B. subtilis IolR might be 2-deoxy-5-keto-D-gluconic acid 6-phosphate [compound (5) in Fig. 1
]. Further studies to prove the above hypothesis are in progress.
 |
ACKNOWLEDGEMENTS
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We wish to thank D. Aoyama, Y. Fujii, S. Goto, H. Hara, Y. Ihori, I. Ishio, S. Inoue, J. Kawai and C. Setogawa for their technical assistance. We also thank F. Matsuura, M. Ohta and A. Tonari, Department of Biotechnology, Fukuyama University, for their indispensable contributions in inosose preparation. We are indebted to P. Pujic, M. Pujic and Y. Yamada for critical reading of the manuscript. This work was supported by a Grant-in-Aid for the Encouragement of Young Scientists to K. Yoshida from the Ministry of Education, Science and Sports and Culture of Japan.
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Received 16 September 2003;
revised 10 November 2003;
accepted 11 November 2003.
Copyright © 2004 Society for General Microbiology.