Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M506477200 on December 7, 2005 Originally published In Press as doi:10.1074/jbc.M506477200 on December 2, 2005

J. Biol. Chem., Vol. 281, Issue 5, 2612-2623, February 3, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An addition or correction has been published
Right arrow All Versions of this Article:
281/5/2612    most recent
M506477200v2
M506477200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Watanabe, S.
Right arrow Articles by Makino, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Watanabe, S.
Right arrow Articles by Makino, K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Cloning, Expression, and Characterization of Bacterial L-Arabinose 1-Dehydrogenase Involved in an Alternative Pathway of L-Arabinose Metabolism*

Seiya Watanabe{ddagger}§||, Tsutomu Kodak§||, and Keisuke Makino§||1

From the {ddagger}Faculty of Engineering, Kyoto University, Kyotodaigakukatsura, Saikyo-ku, Kyoto 615-8530, §Institute of Advanced Energy, Kyoto University, Gokasyo, Uji, Kyoto 611-0011, International Innovation Center, Kyoto University, Yoshidahonmachi, Sakyo-ku, Kyoto 606-8501,and||CREST, Japan Science and Technology Agency, Gokasyo, Uji, Kyoto 611-0011, Japan

Received for publication, June 14, 2005 , and in revised form, November 28, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Azospirillum brasiliense converts L-arabinose to {alpha}-ketoglutarate via five hypothetical enzymatic steps. We purified and characterized L-arabinose 1-dehydrogenase (EC 1.1.1.46 [EC] ), catalyzing the conversion of L-arabinose to L-arabino-{gamma}-lactone as an enzyme responsible for the first step of this alternative pathway of L-arabinose metabolism. The purified enzyme preferred NADP+ to NAD+ as a coenzyme. Kinetic analysis revealed that the enzyme had high catalytic efficiency for both L-arabinose and D-galactose. The gene encoding L-arabinose 1-dehydrogenase was cloned using a partial peptide sequence of the purified enzyme and was overexpressed in Escherichia coli as a fully active enzyme. The enzyme consists of 308 amino acids and has a calculated molecular mass of 33,663.92 Da. The deduced amino acid sequence had some similarity to glucose-fructose oxidoreductase, D-xylose 1-dehydrogenase, and D-galactose 1-dehydrogenase. Site-directed mutagenesis revealed that the enzyme possesses unique catalytic amino acid residues. Northern blot analysis showed that this gene was induced by L-arabinose but not by D-galactose. Furthermore, a disruptant of the L-arabinose 1-dehydrogenase gene did not grow on L-arabinose but grew on D-galactose at the same growth rate as the wild-type strain. There was a partial gene for L-arabinose transport in the flanking region of the L-arabinose 1-dehydrogenase gene. These results indicated that the enzyme is involved in the metabolism of L-arabinose but not D-galactose. This is the first identification of a gene involved in an alternative pathway of L-arabinose metabolism in bacterium.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
L-Arabinose is a major constituent of some plant materials (1), and L-arabinose catabolism is therefore relevant for microorganisms using plant material as a carbon source. The metabolic pathway from L-arabinose to D-xylulose 5-phosphate in bacterium (Fig. 1A) has been investigated extensively. Many bacteria, including Escherichia coli, depend on protein products of the araBAD operon, which contains araB (ribulokinase, EC 2.7.1.16 [EC] ), araA (L-arabinose isomerase, EC 5.3.1.4 [EC] ), and araD (L-ribulose-phosphate 4-epimerase, EC 5.1.3.4 [EC] ) to convert L-arabinose to D-xylulose 5-phosphate through L-ribulose and L-ribulose 5-phosphate (2). Richard et al. (3–5) recently reported a complete fungal pathway (Fig. 1B) containing the sequential action of four oxidoreductases. In this pathway, NAD(P)H-dependent aldose reductase (EC 1.1.1.21 [EC] ) produces L-arabinitol; L-arabinitol 4-dehydrogenase (EC 1.1.1.12 [EC] ) produces L-xylulose; L-xylulose reductase (EC 1.1.1.10 [EC] ) produces D-xylitol; and D-xylulose reductase (EC 1.1.1.9 [EC] ) produces D-xylulose. D-Xylulose is then phosphorylated by xylulokinase (EC 2.7.1.17 [EC] ) to yield D-xylulose 5-phosphate.

It is believed that there are two alternative pathways for bacterial L-arabinose metabolism, which do not involve a phosphorylation reaction, in contrast to the known bacterial and fungal pathways (6–14). In the first pathway, L-arabinose is oxidized to L-arabino-{gamma}-lactone by NAD(P)+-dependent dehydrogenase. The lactone is cleaved by a lactonase to L-arabonate, followed by two successive dehydration reactions forming L-2-keto-3-deoxyarabonate (L-KDA)2 and {alpha}-ketoglutaric semialdehyde. The last step is the NAD(P)+-dependent dehydrogenation of the semialdehyde to {alpha}-ketoglutaric acid. The second pathway has the same initial three steps, but L-KDA is cleaved through an aldolase reaction to glycoaldehyde and pyruvate. No gene-encoding enzyme involved in these alternative pathways of L-arabinose metabolism has been identified so far.

Dehydrogenases for D-arabinose, D-glucose, D-xylose, and D-galactose are known in many organisms. D-Arabinose 1-dehydrogenase (EC 1.1.1.117 [EC] ) is involved in the biosynthesis of D-erythroascorbic acid (15). Glucose 1-dehydrogenase (GDH) is classified into two types, pyrrolo-quinoline-quinone-dependent GDH (EC 1.1.5.2 [EC] ) and NAD(P)-dependent GDH. According to primary structure analysis, these GDH types are not related. NAD(P)-dependent GDH is further classified into two different protein families (16). One GDH (EC 1.1.118 (119)) belongs to a medium chain dehydrogenase/reductase family, which contains an active zinc ion (17); the other GDH is glucose-fructose oxidoreductase (GFOR, EC 1.1.99.28 [EC] ), which catalyzes the coupled intermolecular oxidation-reduction of D-glucose and D-fructose (18–20). GFOR belongs to the Gfo/Idh/MocA family, which also contains D-xylose 1-dehydrogenase (EC 1.1.1.175 [EC] (179)) and D-galactose 1-dehydrogenase (EC 1.1.1.48 [EC] (120)) (21, 22). GDH is involved in the nonphosphorylative Entner-Doudoroff (ED) pathway of Archaea (17, 23–25) and Aspergillus fungi (26) (Fig. 1D). GDH also functions as "gluconolactonase" in this pathway and converts D-glucose to D-gluconate via D-glucono-{delta}-lactone. It is interesting that enzymes of the nonphosphorylative ED pathway are used for the metabolism of both D-glucose and D-galactose in archaeal Sulfolobus solfataricus (17, 27, 28), considering that the alternative pathway of bacterial L-arabinose metabolism seems to be equivalent to the nonphosphorylative ED pathway (Fig. 1, C and D).


Figure 1
View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 1.
A–C, known and proposed pathways of L-arabinose metabolism. A, known bacterial pathway. B, fungal pathway. C, alternative pathway proposed by Novick and Tyler (13) for A. brasiliense (first pathway). L-KDA is also converted to pyruvate and glycolaldehyde in several bacteria (second pathway). D, nonphosphorylative ED pathway.

 
In this study, we focused on a proposed NAD(P)+-dependent dehydrogenase that converts L-arabinose to L-arabino-{gamma}-lactone (referred to as L-arabinose 1-dehydrogenase, EC 1.1.1.46 [EC] ) (Fig. 1C). It has been reported that Azospirillum brasiliense, a bacterium used in this study, can grow on L-arabinose as a sole carbon source and has the first alternative pathway of L-arabinose metabolism (13). This is the first enzymological and molecular biological analysis of the proposed pathway of L-arabinose metabolism.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strain, Culture Conditions, and Preparation of Cell-free Extracts—A. brasiliense ATCC29145 was purchased from RIKEN BioResource Center (Saitama, Japan) and cultured aerobically with vigorously shaking at 30 °C for 24 h in a minimal medium (13) (pH 6.8), containing 4.0 g of KH2PO4, 6.0 g of K2HPO4, 0.2 g of MgSO4·H2O, 0.1 g of NaCl, 0.026 g of CaSO4·2H2O, 1.0 g of NH4Cl, 0.01 g of FeCl3·6H2O, 0.002 g of NaMoO4·2H2O, and 0.0001 g of biotin/liter supplemented with 37 mM L-arabinose. L-Arabinose was sterilized separately by filtration and added to the medium. The grown cells were harvested by centrifugation at 30,000 x g for 20 min, washed with 20 mM potassium phosphate buffer (pH 7.0), containing 2 mM MgCl2 and 10 mM 2-mercaptoethanol (referred to as Buffer A), and stored at -35 °C until used. The washed cells were suspended in Buffer A, disrupted by sonication for 20 min with appropriate intervals on ice using ASTRASON® Ultrasonic Liquid Processor XL2020 (Misonix Inc., New York), and then centrifuged at 108,000 x g for 20 min at 4 °C to obtain cell-free extracts.

PAGE—SDS-PAGE was performed as described by Laemmli (29). Nondenaturing PAGE was performed by omitting SDS and 2-mercaptoethanol from the solution used in SDS-PAGE. Proteins on the gel were stained with Coomassie Brilliant Blue R-250 and destained with 7.5% (v/v) acetic acid in 25% methanol.

Enzyme Activity Assay—L-Arabinose 1-dehydrogenase activity was assayed routinely in the direction of L-arabinose oxidation by measuring the reduction of NAD(P)+ at 340 nm at 30 °C using Jasco spectrophotometer model V-550 (Japan Spectroscopic Co., Ltd., Tokyo, Japan). The standard assay mixture contained 10 mM L-arabinose in 100 mM Tris-HCl (pH 9.0) buffer. The reaction was started by the addition of 10 mM NAD(P)+ solution (100 µl) with a final reaction volume of 1 ml. The kinetic parameters, Km and kcat values, were calculated by Lineweaver-Burk plot. Protein concentrations were determined by the method of Lowry et al. (30) with bovine serum albumin as a standard.

Zymogram Staining Analysis—The cell-free extracts or purified enzyme were separated on nondenaturing PAGE with 12% gel at 4 °C. The gels were then soaked in 10 ml of staining solution (31) consisting of 100 mM Tris-HCl (pH 9.0), 100 mM L-arabinose, 0.25 mM nitro blue tetrazolium, 0.06 mM phenazine methosulfate, and 1 mM NAD(P)+ at room temperature for 15 min. The dehydrogenase activity appeared as a dark band.

Purification of L-Arabinose 1-Dehydrogenase—All purification steps were performed below 4 °C. The cell-free extracts were fractionated between 50 and 60% saturation of (NH4)2SO4. The precipitate was dissolved in a small volume of Buffer A, and the solution was then dialyzed against a large volume of Buffer A containing 1.3 M (NH4)2SO4 overnight. All chromatography was carried out using an {Delta}KTA purifier system (Amersham Biosciences). After insoluble materials were removed by centrifugation, the supernatant was applied to a HiPrep 16/10 Butyl FF column (1.6 x 10 cm, Amersham Biosciences) equilibrated with Buffer A containing 1.3 M (NH4)2SO4 and washed with the same buffer. Proteins were eluted using a reversed linear gradient of 1.3–0 M (NH4)2SO4 in Buffer A (300 ml). The fractions with high enzymatic activity were pooled and dialyzed overnight against a large volume of Buffer A. The enzyme solution was loaded onto a column of HiPrep 16/10 Q FF (1.6 x 10 cm, Amersham Biosciences) equilibrated with Buffer A and washed thoroughly with the same buffer. The column was developed with 300 ml of linear gradient 0–1 M NaCl in Buffer A. The fractions containing L-arabinose 1-dehydrogenase activity were combined and dialyzed against a large volume of 5 mM potassium phosphate (pH 7.0), containing 10 mM 2-mercaptoethanol (referred to as Buffer B) overnight. The enzyme solution was applied to a column of Ceramic Hydroxyapatite Type I (1.6 x 5 cm, Bio-Rad), equilibrated with Buffer B. The column was washed thoroughly with the same buffer and developed with 200 ml of linear gradient 0.005–0.5 M potassium phosphate in Buffer B. The fractions with high enzymatic activity were combined and concentrated by ultrafiltration with Centriplus YM-30 (Millipore) at 18,000 x g for ~2 h. The enzyme solution was loaded onto a column of HiLoad 16/60 Superdex 200 pg column (1.6 x 60 cm, Amersham Biosciences) equilibrated with Buffer A. The active fractions were pooled, concentrated, and re-loaded onto the same column. Proteins in the fractions containing high activity L-arabinose 1-dehydrogenase were analyzed by SDS-PAGE, and the fractions containing a single protein were collected, concentrated, dialyzed against Buffer C (100 mM Tris-HCl (pH 9.0), containing 2 mM MgCl2, 10 mM L-arabinose, 1 mM dithiothreitol, and 50% (v/v) glycerol), and stored at -35 °C until used.

The native molecular mass of L-arabinose 1-dehydrogenase was estimated by gel filtration. The purified enzyme was loaded onto a HiLoad 16/60 Superdex 200 pg column equilibrated with 20 mM potassium phosphate (pH 7.0), containing 2 mM MgCl2 and 1 mM dithiothreitol. High and low molecular weight gel filtration calibration kits (Amersham Biosciences) were used as molecular markers.

Product Identification by HPLC—The product of the dehydrogenation reaction of L-arabinose was identified by HPLC with a Multistation LC-8020 model II system (Tosoh). L-Arabino-{gamma}-lactone was chemically synthesized with boiling potassium arabonate in 0.2 M HCl for 5 min. Potassium arabonate was prepared by the hypoiodite-in-methanol oxidization of L-arabinose (32). A solution containing 100 mM Tris-HCl (pH 9.0), 10 mM L-arabinose, 10 mM NADP+, and the purified enzyme (10 µg) was incubated for 30 min at 30 °C, and 100 µl of this solution was then analyzed. Samples were applied at 30 °C into an Aminex HPX-87H Organic Analysis column (300 x 7.8 mm, Bio-Rad) linked to an RID-8020 refractive index detector (Tosoh) and eluted with 5 mM H2SO4 at a flow rate of 0.6 ml/min.

Determination of N-terminal and Internal Amino Acid Sequences—To determine the N-terminal amino acid sequence of L-arabinose 1-dehydrogenase, the purified enzyme was separated by SDS-PAGE with 12% (w/v) gel, and then transferred to HybondTM-P (Amersham Biosciences) at 3 mA/cm2 for 0.5 h in a transfer buffer (10 mM CAPS (pH 11) containing 10% (v/v) methanol) with a horizontal electrophoretic blotting system (model AE-7500, Atto). After staining and destaining the protein, an area of the membrane corresponding to the protein band of L-arabinose 1-dehydrogenase was excised and analyzed with a ProciseTM 492 HT protein sequencer (Applied Biosystems).

Chemical digestion with cyanogen bromide (CNBr) was carried out to determine internal amino acid sequences (33). The purified L-arabinose 1-dehydrogenase (100 µg) was dialyzed overnight against deionized water and lyophilized. The enzyme protein was digested chemically at room temperature in 70% (v/v) formic acid containing 1% (w/v) CNBr (100 µl) in the dark and under N2 overnight. The solution was diluted with 900 µl of deionized water, frozen with liquid N2, and lyophilized. The sample was dissolved in SDS-PAGE sample buffer (500 mM Tris-HCl (pH 6.8), containing 5% (w/v) SDS, 10% (v/v) glycerol, 0.25% (w/v) bromphenol blue, and 5% (v/v) 2-mercaptoethanol) and separated by SDS-PAGE with 18% (w/v) gel. Peptide fragments on the gel were transferred to a HybondTM-P membrane as described above. After staining and destaining, areas of the membrane corresponding to the two peptide fragments from the L-arabinose 1-dehydrogenase (see in Fig. 2B) were excised and sequenced.

Cloning of L-Arabinose 1-Dehydrogenase Gene—The N-terminal and internal peptide sequences were used to design PCR primers for amplification of a partial DNA fragment of the L-arabinose 1-dehydrogenase gene. Eight upstream primers (U1–U8, 26-mer) were designed from (M)SDQVSLGV, the N-terminal amino acid sequence, as follows: 5'-ATG(TCN/AGY)GAYCARGTN(TCN/AGY) (CTN/TTR)GGNGT-3'. Two downstream primers (D1 and D2, 26-mer) were designed from the internal amino acid sequence (M)LEKPPGAT, as follows: 5'-GTNGCNCCNGGNGGYTTYTC(NAG/YAA)CAT-3'. A. brasiliense genomic DNA was prepared using a DNeasyTM tissue kit (Qiagen). PCR was carried out using a PCR Thermal Cycler-Personal (Takara) for 30 cycles in a 50-µl reaction mixture containing 10 pmol of primers, 1.25 units of Ex Taq® DNA polymerase (Takara), and 300 ng of A. brasiliense genomic DNA under the following conditions: denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, and extension at 72 °C for 30 s, each for 30 cycles. Based on the results of genomic PCR using each set of primers, U6 and D1 were chosen for cloning. The sequences of U6 and D1 were 5'-ATGAGYGAYCARGTNTCNTTRGGNGT-3' and 5'-GTNGCNCCNGGNGGYTTYTCNAGCAT-3', respectively. A single PCR product with a length of ~300 bp was purified, cloned into a pGEM®-T vector (Promega) (referred to as pGEM1), and sequenced using a Dual CyDyeTM terminator sequencing kit (Veritas) and appropriate primers with Long-Read Tower, UBC DNA sequencer (Amersham Biosciences). The inserted fragment was amplified with U6 and D1 primers and with pGEM1 as a template DNA, and the PCR product was purified and utilized as a probe for Southern and Northern blot analysis and colony hybridization (34).

For Southern blot analysis, ~1.8 µg of A. brasiliense genomic DNA was digested with six restriction enzymes, EcoRI, HindIII, NotI, PstI, SalI, and XbaI, separated on 1% (w/v) agarose gel and blotted to HybondTM-N (Amersham Biosciences) by capillary transfer using 10x SSC as a transfer buffer (1x SSC is 15 mM sodium citrate (pH 7.0), and 0.15 M NaCl). The blotted filter was cross-linked in an UV cross-linker CX-2000 (Ultra-Violet Products, Ltd.). A double-stranded probe DNA was labeled with digoxigenin-11-dUTP and hybridized using a DIG-High Prime DNA labeling and detection starter kit (Roche Applied Science). Membrane was visualized using a nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent detection system (Roche Applied Science).

A. brasiliense partial genomic library was prepared with genomic DNA with NotI based on the results of Southern blot analysis. The DNA fragments corresponding to a positive band in size (~2.0 kbp of the length) were ligated to a plasmid pBluescript® SK(-) (Stratagene). Colony hybridization was carried out under the same conditions as Southern blot analysis except for the use of nylon membranes for colony and plaque hybridization (Roche Applied Science). The plasmid from a positive clone (referred to as pBS1) was purified, and the inserted A. brasiliense genome fragment was sequenced.

Northern Blot Analysis—A. brasiliense cells were cultured at 30 °C to the mid-log phase (A600 = 0.6–0.8) in minimal medium supplemented with 37 mM appropriate sugar (D-glucose, L-arabinose, D-galactose, or D-xylose) or nutrient medium (10 g of peptone, 10 g of meat extract, and 5.0 g of NaCl (pH 7.0–7.2)) and harvested by centrifugation. Total RNAs from A. brasiliense were prepared with an RNeasy® mini kit (Qiagen) and subsequently treated with RNase-free DNase I. The isolated RNA (4 µg) was subjected to electrophoresis on 1.2% (w/v) agarose gel containing 0.66 M formaldehyde. The subsequent steps were performed using the same methods as for Southern blot analysis.

Cloning of the L-Arabinose 1-Dehydrogenase Gene into Expression Plasmid Vector—To introduce the restriction site for BglII at the 5'-end and PstI at the 3'-end of the L-arabinose 1-dehydrogenase gene, PCR was carried out using pBS1 as a template and the following two primers (lowercase letters indicate additional bases for introducing digestion sites of BglII and PstI (underlined letters)): 5'-caccatagaTCTGATCAGGTTTCGCTGGGTG-3' (HISBglII) and 5'-gcttggctgcagTCAGCGGCCGAACGCGTCG-3' (HISPstI). The amplified DNA fragment was introduced into BamHI-PstI sites in pQE-80L (Qiagen), a plasmid vector for conferring N-terminal His6 tag on the expressed proteins, to obtain pHISWT.

Site-directed Mutagenesis—The following sense and antisense primers were designed to introduce the mutations into the L-arabinose 1-dehydrogenase gene (the mutated regions are underlined): to substitute Ala for Asp168 (D168A), 5'-CGGCGTGTTCGCGCCGGGCATC-3' (D168AS) and 5'-GATGCCCGGCGCGAACACGCCG-3' (D168AAS); to substitute Ala for Asn172 (N172A), 5'-CCCGGGCATCGCGGCGCTGTCG-3' (N172AS) and 5'-CGACAGCGCCGCGATGCCCGGG-3' (N172AAS). The mutations were introduced by sequential steps of PCR (35) with small modifications. In the first round, two reactions, I and II, were performed with the appropriate primers and pHISWT as a template: reaction I, HISBglII and one of the antisense primers containing the mutations; and reaction II, one of the sense primers containing the mutations and HISPstI. In the final amplification step, purified overlapping PCR products were used as templates and HISBglII and HISPstI as primers. The final PCR products were cloned into pQE-80L to obtain plasmids pHISD168A and pHISN172A, respectively. The coding region of the mutated genes was confirmed by subsequent sequencing in both directions.

Functional Expression and Purification of His6-tagged L-Arabinose 1-Dehydrogenase—E. coli DH5{alpha} harboring the expression plasmid for the His6-tagged wild-type and mutated enzymes was grown at 37 °C to a turbidity of 0.6 at 600 nm in Super Broth medium (12 g of tryptone, 24 g of yeast extract, 5 ml of glycerol, 3.81 g of KH2PO4, and 12.5 g of K2HPO4/liter (pH 7.0)) containing 50 mg/liter ampicillin. After the addition of 1 mM of isopropyl-beta-D-thiogalactopyranoside, the culture was further grown for 6 h to induce the expression of His6-tagged L-arabinose 1-dehydrogenase protein. Cells were harvested and resuspended in Buffer D (50 mM sodium phosphate containing 2 mM MgCl2, 300 mM NaCl, 1 mM L-arabinose, 10 mM 2-mercaptoethanol, and 10 mM imidazole (pH 8.0)). The cells were then disrupted by sonication, and the solution was centrifuged. The supernatant was loaded onto a nickel-nitrilotriacetic acid spin column (Qiagen) equilibrated with Buffer D. The column was washed three times with Buffer E (Buffer D containing 10% (v/v) glycerol and 50 mM imidazole instead of 10 mM imidazole (pH 8.0)). The enzymes were then eluted with Buffer F (Buffer E containing 250 mM imidazole instead of 50 mM imidazole (pH 8.0)). The elutant was dialyzed against Buffer C and stored at -35 °C until use.

Western Blot Analysis of His6-tagged L-Arabinose 1-Dehydrogenase—For Western blot analysis, the purified L-arabinose 1-dehydrogenase from A. brasiliense and/or recombinant His6-tagged L-arabinose 1-dehydrogenase from E. coli was separated by SDS-PAGE, and the proteins on the gels were transferred onto a nitrocellulose membrane (HybondTM-ECL; Amersham Biosciences). Western blot analysis was carried out using the ECLTM Western blotting analysis system (Amersham Biosciences) and RGS·His horseradish peroxidase antibody, a horseradish peroxidase-fused mouse monoclonal antibody against Arg-Gly-Ser-His6 in the N-terminal additional peptide of the expressed recombinant proteins (Qiagen).


Figure 2
View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 2.
PAGE of L-arabinose 1-dehydrogenase. A, SDS-PAGE of purification. M is marker proteins. Lane 1, cell-free extracts (50 µg); lane 2, ammonium sulfate fractionation (50 µg); lane 3, HiPrep 16/10 Butyl FF (10 µg); lane 4, HiPrep 16/10 Q FF (10 µg); lane 5, CHT ceramic hydroxyapatite (10 µg); lane 6, HiLoad 16/60 Superdex 200 pg 1st (10 µg); lane 7, HiLoad 16/60 Superdex 200 pg 2nd (10 µg). B, SDS-PAGE of CNBr digestion. The purified enzyme (100 µg) was digested chemically with CNBr, and the solution corresponding to 5 µg of protein was applied. Two observed bands except undigested polypeptide were referred to as Fragments I and II with molecular masses of 26.4 and 0.92 kDa, respectively. C, nondenaturing PAGE (lane 1) and zymogram-staining analysis (lanes 2–5). The purified enzyme of 5 µg and cell-free extracts of 100 µg were applied. NAD+ and NADP+ in lanes 2 and 4 and lanes 3 and 5, respectively, were used as a coenzyme.

 
Disruptant Construction—The overall scheme of the plasmid construction for disruption of the L-arabinose 1-dehydrogenase gene is shown in Fig. 9A. The Tn5-derived 1.3-kb BamHI kanamycin resistance (Kmr) cassette of pUC4K (Amersham Biosciences) was inserted into the single BamHI site in the coding sequence of the L-arabinose 1-dehydrogenase gene of pHISWT to yield pHISWT::Km. To introduce the restriction site for MfeI at the 5'- and 3'-end of the DNA fragment containing the Kmr gene in the L-arabinose 1-dehydrogenase gene, PCR was carried out using pHISWT::Km as a template and the following two primers (lowercase letters indicate additional bases for introducing digestion sites of MfeI (underlined letters)): 5'-caccatcaattgGATCAGGTTTCGCTGGGTGTCGTCGGCATCG-3' (MfeI-up) and 5'-gcttggcaattgTCAGCGGCCGAACGCGTCGGTCTGCACGCGC-3' (MfeI-down). The 2.3-kbp MfeI DNA fragment was subcloned into the EcoRI site in the chloramphenicol resistance (Cmr) cassette of the suicide vector pSUP202 (36) to yield pSUPWT::Km.

E. coli S17-1 (36) was transformed with pSUPWT::Km, and then the transformant was further mobilized to A. brasiliense by biparental mating. The transconjugants were selected on a minimal medium agar plate supplemented with 5 g of sodium malate and 25 µg of kanamycin/liter using Kmr (the presence of Kmr cassette) and TcS (loss of pSUP202) phenotypes. The construction was confirmed by genomic PCR and Southern hybridization on total DNA digested with NotI. One of the resulting disruptants of A. brasiliense was named {Delta}ARA5034 and was used in this study.

Amino Acid Sequence Alignment and Phylogenetic Analysis—Protein sequence of L-arabinose 1-dehydrogenase from A. brasiliense was analyzed using the Protein-BLAST and ClustalW program distributed by DDBJ (www.ddbj.nig.ac.jp). The phylogenetic tree was produced using the TreeView 1.6.1. program.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Purification of L-Arabinose 1-Dehydrogenase from A. brasiliense—NAD+- and NADP+-dependent enzymatic oxidization of L-arabinose was found in cell-free extracts prepared from A. brasiliense cells grown on L-arabinose as a sole carbon source (Fig. 2C). The L-arabinose 1-dehydrogenase was purified by ammonium fractionation and five chromatographic steps. A typical result of purification is summarized in Table 1. During the purification procedure, the ratio of NAD+- and NAD+-linked activity remained almost constant, suggesting the presence of only one protein as L-arabinose 1-dehydrogenase. The purified enzyme was electrophoretically homogeneous (Fig. 2, A and C) and showed NAD+- and NADP+-dependent specific activity of 25 and 44 units/mg protein, respectively (Table 1). SDS-PAGE revealed only one subunit with an apparent value of 39.5 ± 0.7 kDa (Fig. 2A). To estimate native molecular mass, the enzyme was loaded onto a HiLoad 16/60 Superdex 200 pg column. The value from the calibration curve with marker proteins was ~46.4 ± 1.9 kDa (data not shown), indicating that the L-arabinose 1-dehydrogenase is monomeric. There was no significant increase in activity in the presence of MgCl2, MnCl2, ZnCl2, CoCl2, NiCl2, or CaCl2 at final concentrations of 1 mM (data not shown). Zymogram staining analysis showed a major active band in the cell-free extracts. The position corresponded to that of the purified enzyme (Fig. 2C). A minor staining band with NAD+ as a coenzyme may be derived from the concomitant activity of other NAD+-dependent sugar dehydrogenases.


View this table:
[in this window]
[in a new window]
  		TABLE 1
Summary of L-arabinose 1-dehydrogenase purification from A. brasiliense

 


Figure 3
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 3.
Relationship between catalytic efficiency (kcat/Km) and configuration of the substrate in L-arabinose 1-dehydrogenase. The kcat/Km values were taken from the values in Table 2 with NAD+ (light gray bar) and NADP+ (dark gray bar). Asterisks indicate the inactive substrate. Gray-shaded configuration is identical to that of L-arabinose.

 
Substrate Specificity and Kinetic Analysis—In addition to L-arabinose, various sugars were tested as substrates for dehydrogenase. In the first approach, the sugar concentrations were fixed at 10 mM. Activity was observed with L-arabinose, D-galactose, D-xylose, and D-talose but not with D-arabinose, D-glucose, D-ribose, L-xylose, L-mannose, L-lyxose, and D-fructose (less than 1% of the activity with L-arabinose). The enzyme was subjected to further kinetic analysis with the active substrates, and the determined parameters are listed in Table 2. The catalytic efficiency (kcat/Km) values with L-arabinose, D-galactose, and D-xylose in the presence of NADP+ were significantly higher than those in the presence of NAD+ because of the lower values of Km and the higher values of kcat. Furthermore, when L-arabinose was used as a substrate, the enzyme showed 5.6-fold higher affinity with NADP+ (Km = 0.0095 ± 0.0083 mM) than NAD+ (Km = 0.053 ± 0.020 mM). The kinetic parameters of D-galactose were very similar to those of L-arabinose in the presence of either NAD+ or NADP+, indicating that the enzyme functions not only as L-arabinose 1-dehydrogenase but also "D-galactose 1-dehydrogenase." The enzyme showed 51- and 79-fold lower values of kcat/Km with D-talose in the presence of NAD+ and NADP+, respectively, compared with those with L-arabinose. kcat/Km values with D-xylose were lower than those with D-talose by 2 orders of magnitude in the presence of either NAD+ or NADP+ and because of remarkably high Km values and low kcat values.


View this table:
[in this window]
[in a new window]
  		TABLE 2
Kinetic parameters of L-arabinose 1-dehydrogenase Stocked L-arabinose 1-dehydrogenase was dialyzed against 100 mM Tris-HCl (pH 9.0) containing 2 mM MgCl2, overnight at 4 °C before enzyme activity was measured. Values are the means ± S.D., n = 3.

 
Enzyme activities with these sugars, and the stereoconfiguration of these sugars are shown in Fig. 3. Active sugars for L-arabinose 1-dehydrogenase belong not only to pentose(s) but also hexose(s), indicating that the activity is not dependent on the C-5 and C-6 configuration. D-Galactose has the same configuration as L-arabinose at C-2, C-3, and C-4. The activity was also found with D-talose, C-1 epimer of D-galactose, but not with L-mannose and D-glucose, C-3 and C-4 epimer, respectively. Similarly, the activity with D-xylose, the C-4 epimer of L-arabinose, decreased significantly, and no activity was observed with L-lyxose, the C-3 epimer of L-arabinose. It therefore appeared that the enzyme prefers the L-arabinose-specific configuration at C-3 and C-4.


Figure 4
View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 4.
HPLC analysis of the reaction products. L-Arabinose (A), L-arabino-{gamma}-lactone (B), L-arabonate (C), and the enzyme reaction products (D) were loaded into an Aminex HPX-87H Organic Analysis column and detected with a refractive index detector.

 
It has been reported that the product of the enzyme reaction in the cell-free extract system is L-arabino-{gamma}-lactone by paper chromatography (10, 13, 14). We reinvestigated the product of the purified enzyme by HPLC as shown in Fig. 4. The retention time of L-arabino-{gamma}-lactone was slightly earlier than that of L-arabinose, and the main product from L-arabinose was L-arabino-{gamma}-lactone. The minor concomitant L-arabonate is probably due to spontaneous lactone hydrolysis (see Fig. 4B). These results indicated that the enzyme possessed only dehydrogenase activity with L-arabinose but not lactonase with L-arabino-{gamma}-lactone.

Cloning of the Gene Encoding L-Arabinose 1-Dehydrogenase and Its Functional Expression in E. coli—The N-terminal amino acid sequence up to 15 amino acid residues of L-arabinose 1-dehydrogenase was as follows: SDQVSLGVVGIGKIA. To determine the internal amino acid sequence, the purified enzyme was digested chemically by CNBr, which digests specifically at the C termini of methionyl residues in polypeptides. As shown in Fig. 2B, two peptide bands with ~26.4 and 0.92 kDa of molecular mass (referred to as fragments I and II, respectively) were found on SDS-polyacrylamide gel. The 15-amino acid sequence of Fragment II was completely identical to the N-terminal sequence. The amino acid sequence up to 15 amino acid residues of Fragment I was as follows: (M)LEKPPGATLGEVAVL, suggesting that Fragment II is located upstream of Fragment I in the protein sequences. Using synthetic DNA primers designed from N-terminal and internal amino acid sequences, an ~300-bp nucleotide fragment of the L-arabinose 1-dehydrogenase gene was obtained by genomic PCR. Southern blot analysis with this DNA fragment as a probe showed a single band on each restriction enzyme digestion of A. brasiliense genomic DNA with EcoRI, HindIII, NotI, PstI, SalI, and XbaI of ~3.9, 15, 2.0, 4.5, 2.2, and 17 kbp (data not shown), revealing only a single copy of the gene on the genome. Based on the results, we isolated a 1,805-bp NotI fragment containing the full L-arabinose 1-dehydrogenase gene (Fig. 5). The G + C content of the entire sequence was significantly high (~69%), consistent with that of chromosomal DNA from A. brasiliense (70%) (37). The L-arabinose 1-dehydrogenase was 927 bp long, and a putative ribosome-binding site (Shine-Dalgarno sequence, AGGAG) was found 9–13 bases upstream of the ATG codon. The deduced amino acid sequence of the protein (polypeptide of 309 amino acids with a predicted mass of 33,795.12 Da) agreed with the determined N-terminal amino acid sequences from the corresponding purified protein, except for removed formylmethionine. The internal amino acid sequences determined by the digestion of purified protein with CNBr were found at positions 89–103 of the protein sequences. The nucleotide sequence was submitted to GenBankTM with accession number AB211983 [GenBank] . Two partial ORFs were found upstream and downstream of the L-arabinose 1-dehydrogenase gene (referred to as ORF1 and ORF2, respectively) (Fig. 5). The deduced ORF1 and ORF2 proteins showed significant identity with dihydrodipicolinate synthase/N-acetylneuraminate lyase and periplasmic L-arabinose-binding proteins (involving in the ABC-type transport system) from many organisms, respectively.


Figure 5
View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 5.
Comparison of a chromosomal region containing the L-arabinose 1-dehydrogenase gene and its flanking region between A. brasiliense and B. capacia strain R18149 [GenBank] . The NotI-NotI DNA fragment of A. brasiliense genome DNA was cloned in this study.

 
L-Arabinose 1-dehydrogenase was overexpressed in E. coli cells by induction with isopropyl-beta-D-thiogalactopyranoside as a His6-tagged enzyme and purified homogeneously with a nickel-chelating affinity column (Fig. 6A). The mobility of the recombinant enzyme in SDS-PAGE and zymogram-staining analysis (Fig. 6, A and C) was slightly later than that of the native enzyme purified from A. brasiliense cells because of the additional 13 amino acid residues, including a His6 tag at the N terminus. Western blot analysis with anti-His6 tag antibody confirmed the His6 tag in the enzyme expressed in E. coli (Fig. 6B). The enzyme showed similar NADP+ preference and kinetic parameters for L-arabinose in the presence of NAD(P)+ to those of the native enzyme (Tables 2 and 3), confirming that the isolated gene encodes L-arabinose 1-dehydrogenase.


View this table:
[in this window]
[in a new window]
  		TABLE 3
Kinetic parameters of recombinant L-arabinose 1-dehydrogenase Stocked L-arabinose 1-dehydrogenase was dialyzed against 100 mM Tris-HCl (pH 9.0) containing 2 mM MgCl2, overnight at 4 °C. Values are the means ± S.D., n = 3.

 
Amino Acid Sequence Analysis of L-Arabinose 1-Dehydrogenase—Protein-BLAST analysis revealed that L-arabinose 1-dehydrogenase is a putative member of the Gfo/Idh/MocA protein family, which includes GFOR (18–20), D-xylose 1-dehydrogenase (21), dimeric dihydrodiol dehydrogenase (DD) (38), and myo-inositol 2-dehydrogenase (39) (Fig. 7A). The enzyme activity was not influenced by 1 mM EDTA (data not shown), supporting the hypothesis that the enzyme does not belong to a medium chain dehydrogenase/reductase family, which contains an active zinc ion (see Introduction). It is noteworthy that GDH in this family is a bifunctional enzyme with gluconolactonase (17, 23–25), whereas L-arabinose 1-dehydrogenase possesses no lactonase activity with L-arabino-{gamma}-lactone (Fig. 4).

High degrees of similarity to L-arabinose 1-dehydrogenase (over 100 bits of score) were found in bacterial (putative) oxidoreductases and dehydrogenases, including D-galactose 1-dehydrogenase from Pseudomonas fluorescens (22), confirming that L-arabinose 1-dehydrogenase is also a D-galactose 1-dehydrogenase. In the unrooted phylogenetic tree, these enzymes containing L-arabinose 1-dehydrogenase formed a single subfamily (Fig. 7B, Subfamily I). L-Arabinose/D-galactose 1-dehydrogenase activities have been known to show NAD+ preference (10, 13, 14, 40–42). To our knowledge, this study is the first concerning NADP+-preferring L-arabinose and/or D-galactose 1-dehydrogenase.


Figure 6
View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 6.
SDS-PAGE, Western blot, and zymogram analysis of recombinant L-arabinose 1-dehydrogenase. Lane 1, native enzyme purified from A. brasiliense cells; lanes 2–4, His6-tagged recombinant enzymes of wild-type (lane 2), D168A (lane 3), and N172A (lane 4); M, marker proteins. A, SDS-PAGE. Five µg of purified protein was applied on 12% (w/v) SDS-polyacrylamide gel. B, Western blot analysis. One µg of protein was electrophoresed. Western blot analysis was carried out using anti-His6 antibody. C, zymogram staining analysis. One µg of protein was applied. After electrophoresis, the gel was soaked into staining solution in the presence of 100 mM L-arabinose and 1 mM NADP+.

 
Identification of Catalytic Amino Acid Residues—It is known that Asp265 and Tyr269 in GFOR from Zymomonas mobilis (ZmGFOR) (20) and Asp176 and Tyr180 in dimeric DD (43, 44) are important for the catalytic function. It was almost impossible to align the amino acid sequence of L-arabinose 1-dehydrogenase and ZmGFOR in the C-terminal domains because of weak sequential homology. Therefore, we carried out multiple alignments with over 500 amino acid sequences of Gfo/Idh/MocA family enzymes, revealing that Asp168–Asn172 in L-arabinose 1-dehydrogenase corresponds to Asp265–Tyr269 in ZmGFOR and Asp176-Tyr180 in dimeric DD (Fig. 7, A and C). To obtain insight into the catalytic mechanism of L-arabinose 1-dehydrogenase, Asp168 and Asn172 were substituted with alanine residues by site-directed mutagenesis, as described under "Experimental Procedures," to construct D168A and N172A mutants, respectively. They were overexpressed in E. coli cells as a His6-tagged enzyme and purified with the same procedures for the wild-type enzyme (Fig. 6A). No activity of these mutants was found in zymogram-staining analysis (Fig. 6B). Kinetic analysis showed that the N172A mutant decreased by 4 orders of magnitude in kcat/Km values, compared with the wild-type enzyme (Table 3). The D168A mutant showed no activity under standard assay conditions. These results suggested that both Asp168 and Asn172 were important for catalytic function in L-arabinose 1-dehydrogenase.

Expression of L-Arabinose 1-Dehydrogenase in A. brasiliense and Mutant Analysis—L-Arabinose 1-dehydrogenase has been shown to have dehydrogenase activities with other sugars such as D-galactose and D-xylose in vitro (Table 2). To estimate the physiological role of these dehydrogenase activities, Northern blot analysis was carried out with RNAs isolated from A. brasiliense cells grown on different carbon sources as follows: L-arabinose, D-galactose, and D-xylose (active substrates in vitro) and D-glucose (inactive substrate in vitro) (Fig. 8A). The significant expression of the L-arabinose 1-dehydrogenase gene was only found in cells grown on L-arabinose as a sole carbon source. Changes in L-arabinose 1-dehydrogenase activity in the cell-free extracts prepared from cells grown on different carbon sources (Fig. 8, B and C) were analogous to those observed at the level of transcription.

Furthermore, we constructed an A. brasiliense disruptant of the L-arabinose 1-dehydrogenase gene, designated {Delta}ARA5034, by biparental mating and double homologous recombination as described under "Experimental Procedures" (Fig. 9A). When {Delta}ARA5034 was cultivated in a minimal medium supplemented with L-arabinose as a sole carbon source, no substantial growth was found. On the other hand, no difference in the growth rate on D-glucose, D-galactose, and D-xylose was found between the wild-type and {Delta}ARA5034 (Fig. 9B). Northern blot and disruptant analysis revealed that enzymatic activity with L-arabinose in vitro has a physiological meaning, but activity with D-galactose does not, and that the L-arabinose 1-dehydrogenase gene is clearly involved in L-arabinose metabolism of A. brasiliense.


Figure 7
View larger version (55K):
[in this window]
[in a new window]
  		FIGURE 7.
A, multiple sequence alignment of deduced amino acid sequences of L-arabinose 1-dehydrogenase from A. brasiliense and D-galactose 1-dehydrogenase from P. fluorescens and GFOR from Z. mobilis. A 52-amino acid sequence corresponding to a signal peptide in ZmGFOR was omitted. Secondary structures of ZmGFOR are shown on the sequence. In the crystal structure of ZmGFOR, amino acid residues forming a hydrogen bond with the substrate (closed circle) or 2'-phosphate of NADP+ (shaded circle), around the bound substrate within a distance of4Å(up arrow) and functioning as a catalytic base (open circle), are indicated below the sequences. GenBankTM accession numbers for protein sequences are in B. B, the phylogenetic relationships among bacterial L-arabinose/D-galactose 1-dehydrogenases (subfamily I), bacterial GFORs, and archaeal D-xylose 1-dehydrogenase (subfamily II), mammalian dimeric DDs (subfamily III) and bacterial myo-inositol 2-dehydrogenases (subfamily IV). The number on each branch indicates the bootstrap value. C, partial alignment of amino acid sequences around the site-directed mutated region of L-arabinose 1-dehydrogenase with other proteins of the Gfo/Idh/MocA family and G6PDH. Letters in parentheses are the GenBankTM accession number. White letters in black boxes indicate the positions of amino acid residues of L-arabinose 1-dehydrogenase substituted by site-directed mutagenesis in this study. Highly conserved amino acid residues are in boldface letters.

 


Figure 8
View larger version (56K):
[in this window]
[in a new window]
  		FIGURE 8.
Effect of a carbon source on the intercellular expression level of L-arabinose 1-dehydrogenase. A, Northern blot analysis. Total RNAs (4 µg per lane) were isolated from A. brasiliense cells grown in nutrient medium (lane 1) and synthetic medium containing L-arabinose (lane 2), D-galactose (lane 3), D-xylose (lane 4), and D-glucose (lane 5) at concentrations of 37 mM. B, zymogram-staining analysis using cell-free extracts. Fifty µg of protein was applied. After electrophoresis, the gel was soaked into staining solution in the presence of 100 mM L-arabinose and 1 mM NADP+. N is a purified native L-arabinose 1-dehydrogenase (1 µg). C, enzyme activity in cell-free extracts. L-Arabinose 1-dehydrogenase activity was measured under standard assay conditions using NADP+ as a coenzyme. Values are the means ± S.D., n = 3.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we reported a novel L-arabinose 1-dehydrogenase involved in an alternative pathway of L-arabinose metabolism. The purified enzyme showed significant NADP+-preferring activity not only with L-arabinose but also D-galactose. The gene expression was only induced by L-arabinose, suggesting that the physiological role is limited in L-arabinose metabolism. L-Arabinose 1-dehydrogenase was a potential member of the Gfo/Idh/MocA protein family and contained some unique catalytic amino acid residues.

Catalytic Insight into L-Arabinose 1-Dehydrogenase—Generally, subunit structures of the Gfo/Idh/MocA family are comprised of two domains, the N-terminal and C-terminal domains. The N-terminal domain (~120 amino acid residues) has a characteristic GXGXX(G/A) fingerprint motif (45) in the classical beta{alpha}beta dinucleotide binding pocket (46) and a recently postulated fingerprint motif for a novel class of dehydrogenases with a consensus AGKHVXCEKP (where X is any amino acid) (19). These motifs are also conserved in L-arabinose 1-dehydrogenase with little deviation (Fig. 7A, motifs I and II). The C-terminal domain of Gfo/Idh/MocA family enzymes (~220 amino acid residues) shows significant sequential divergence and contains some essential amino acid residues for enzyme catalysis and/or substrate binding (Fig. 7A, open and closed circles, respectively), probably because of inherent substrate specificity in each enzyme. The structural features and catalytic mechanisms of ZmGFOR have been extensively studied by enzymatic and crystallographic analysis (18–20). The enzyme contains tightly bound coenzyme NADP+, and the strict NADP+ specificity is rationalized by Ser116, Lys121, and Tyr139 (Fig. 7A, shaded circle) (47). In particular, the serine residue is important to distinguish between NAD and NADP+ with NAD+; the S116D mutant of ZmGFOR shows significant activity+, and NAD+-dependent myo-inositol 2-dehydrogenase possesses aspartate residue at the corresponding position (19). On the other hand, NAD+-preferring D-galactose 1-dehydrogenase from P. fluorescens (22, 41) possesses threonine residue but not aspartate at the corresponding position to Ser116. No L-arabinose/D-galactose 1-dehydrogenase has amino acid residues equivalent to Lys121 and Tyr139. These comparisons indicate that there are several modifications for determining the coenzyme specificity between L-arabinose/D-galactose 1-dehydrogenases and other Gfo/Idh/MocA family enzymes. Furthermore, compared with ZmGFOR, almost all putative amino acid residues around the substrate binding regions are substituted for those with functionally different characteristics in L-arabinose 1-dehydrogenase (Fig. 7A, up arrow), clearly creating a different environment at the active center.

A Novel Catalyst Type in L-Arabinose 1-Dehydrogenase—Glucose-6-phosphate dehydrogenase (G6PDH) is a structurally homologous enzyme with GFOR and has a very strong functional relationship with GFOR. In Leuconostoc mesenteroides G6PDH (LmG6PDH) (48, 49), the catalytic base (His240) removes a proton from the C-1 OH group of Glc-6-P, and NAD(P)+ abstracts a hydride ion (H-) from the C-1 atom. Asp235 forms a hydrogen bond with C-2 OH of Glc-6-P. In ZmGFOR, the structurally homologous Asp265–Tyr269 residues play equivalent roles (Fig. 7, A and C) (20). In enzymes of the Gfo/Idh/MocA family, the pair of Asp-(Tyr/His) is conserved completely (Fig. 7C). Multiple alignments revealed that Asp168–Asn172 in L-arabinose 1-dehydrogenase corresponds to Asp265–Tyr269 in ZmGFOR and Asp235–His240 in LmG6PDH (Fig. 7, A and C). It is very interesting to identify the involvement of Asn172 in the catalytic function because the LmG6PDH H240N mutant shows a decreased kcat value by 4 orders of magnitude (48) and a 10-fold higher Km value for Glc-6-P. Therefore, we constructed two mutants of L-arabinose 1-dehydrogenase, D168A and N172A, by site-directed mutagenesis. When Asp168 was replaced with Ala, a D168A mutant of L-arabinose 1-dehydrogenase lacked sufficient activity for detection in our standard assay conditions and zymogram staining analysis (Table 3 and Fig. 6C). On the other hand, the N172A mutant retained some enzymatic activity (less than 1% of kcat/Km of the wild-type enzyme) (Table 3), whereas the activity was too weak to detect in zymogram staining analysis (Fig. 6C). These results suggest that Asn172 has a very important role in catalysis but is not absolutely necessary. This site-directed mutagenesis indicates that Asp168 and Asn172 are surely involved in the catalytic function of L-arabinose 1-dehydrogenase, raising doubt as to whether Asn172 is a catalytic base. In the crystal structure of wild-type LmG6PDH, the distance between Asp235 and C-1 OH of Glc-6-P is 4.8 Å, which is only slightly distant for a hydrogen bond (49). Considering that Asp168 in L-arabinose 1-dehydrogenase corresponds to Asp235 in LmG6PDH, we speculate that Asp168 functions as a catalytic base in L-arabinose 1-dehydrogenase. This novel mechanism evolved early from an ancestral Gfo/Idh/MocA enzyme possessing a pair of Asp-(Tyr/His), such as ZmGFOR, because the asparagine residue equivalent to Asn172 in L-arabinose 1-dehydrogenase is only found in the enzymes of subfamily I (Fig. 7, B and C).

Physiological Insight of L-Arabinose Metabolism in A. brasiliense—We could find many L-arabinose 1-dehydrogenase gene homologs on the bacterial genome sequence (Fig. 7B, Subfamily I). It is noteworthy that all members are from bacteria associating pathologically and/or parasitically with plants. In particular, the nucleotide sequences of the L-arabinose 1-dehydrogenase gene and the flanking region of A. brasiliense show high similarity (~91%) to part of the genomic sequence of Burkholderia cepacia strain R18149 [GenBank] (Fig. 5), which was isolated from forest soil. On the other hand, the comparison of rRNA sequences revealed their far phylogenic relationship (~77% of identity) (data not shown). These results suggest that wide range genome transportation among plant-associated bacteria occurred at a very early evolutional stage.


Figure 9
View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 9.
A, schematic diagram of the plasmid construction for disruption of the L-arabinose 1-dehydrogenase gene. Gray-colored region indicates the region of the L-arabinose 1-dehydrogenase gene. B, E, and M indicate BamHI, EcoRI, and MfeI restriction enzyme sites, respectively. Kmr, Tcr, Ampr, and Cmr are kanamycin, tetracycline, ampicillin, and chloramphenicol resistance cassettes, respectively. B, growth of wild-type and {Delta}ARA5034 strain of A. brasiliense on D-glucose ({circ}), L-arabinose ({square}), D-galactose ({Delta}), and D-xylose (224) as a sole carbon source. Each sugar was supplemented at the concentration of 37 mM in a minimal medium. For {Delta}ARA5034, 25 µg of kanamycin/liter was added. Cell growth was monitored by measuring absorbance at 600 nm.

 
The analysis of substrate specificity revealed that the purified enzyme functions not only as L-arabinose 1-dehydrogenase but also D-galactose 1-dehydrogenase. However, gene induction was only observed in A. brasiliense cells grown on L-arabinose (Fig. 8), and {Delta}ARA5034, a disruptant of the L-arabinose 1-dehydrogenase gene, showed the same growth rate in D-galactose as the wild-type (Fig. 9B). A deduced ORF2 protein, located downstream of the L-arabinose 1-dehydrogenase gene (Fig. 5), has a high degree of sequential similarity to bacterial periplasmic L-arabinose-binding proteins, which belong to ATP-binding cassette (ABC)-type sugar transporters. Generally, this type of bacterial transporter consists of a periplasmic substrate-binding protein, an ATP-binding complex (ATPase), and a membrane-spanning protein (permease). On the genome of B. cepacia strain R18149 [GenBank] , the corresponding three genes are clustered downstream of the putative L-arabinose 1-dehydrogenase gene (Fig. 5). This may also appear in A. brasiliense, considering the sequence similarity of these two organisms. The enzymatic and genetic evidence indicates that the L-arabinose 1-dehydrogenase gene is only involved in the metabolic pathway of L-arabinose and not D-galactose. Recently, Moore et al. (50) reported that two homologous genes to a putative transcriptional regulator (ZP_00218288) and dehydrogenase (ZP_00218291) of B. cepacia strain R18149 [GenBank] (Fig. 5) are absolutely necessary for growth on L-arabinose in Burkholderia thiailandensis, a close relative bacteria of B. cepacia strain R18149 [GenBank] ; deletion mutants of one of these two genes showed an L-arabinose-negative phenotype as well as {Delta}ARA5034 (Fig. 9B). It should be noted that these two Burkholderia strains possess the same L-arabinose metabolic pathway as A. brasiliense, and the clustered genes, including L-arabinose 1-dehydrogenase, comprise a transcriptional unit.

Van Bastelaere et al. (51) identified a plant root exudate-inducible 40-kDa protein from A. brasiliense (designated as SbpA (sugar-binding protein A)). The deduced amino acid sequences are very similar to virulent ChvE protein from Agrobacterium tumefaciens, a putative periplasmic ABC-type transporter (52). The SbpA protein is involved in the uptake of D-galactose, L-arabinose, and D-fructose, whereas residual uptake of these sugars is found in the sbpA mutant. These findings suggest the existence of multiple transport systems in A. brasiliense. Because the deduced ORF2 protein from A. brasiliense shows no similarity to SbpA, it can be speculated that this protein is an alternative L-arabinose transporter(s). Further investigation of the region surrounding the L-arabinose 1-dehydrogenase gene is important for the elucidation of this alternative L-arabinose metabolism.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB211983 [GenBank] .

* This work was supported by the Center of Excellence program for the"Establishment of Centers of Excellence on Sustainable Energy System,"a grant-in-aid for scientific research, and grants for regional science and technology promotion from the Ministry of Education, Science, Sports, and Culture, Japan, and by CREST of the Japan Science and Technology Corp. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 81-774-38-3517; Fax: 81-774-38-3524; E-mail: kmak{at}iae.kyoto-u.ac.jp.

2 The abbreviations used are: L-KDA, L-2-keto-3-deoxyarabonate; GDH, glucose 1-dehydrogenase; ED pathway, Entner-Doudoroff pathway; GFOR, glucose-fructose oxidoreductase; CNBr, cyanogen bromide; DD, dihydrodiol dehydrogenase; ZmGFOR, GFOR from Z. mobilis; G6PDH, glucose-6-phosphate dehydrogenase; LmG6PDH, G6PDH from L. mesenteroides; CAPS, cyclohexylaminopropanesulfonic acid; HPLC, high pressure liquid chromatography; ORF, open reading frame; ABC, ATP-binding cassette. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Kiwamu Minamisawa (Tohoku University) and Dr. Masayuki Inui (Research Institute of Innovative Technology for the Earth) for the gifts of the plasmid pSUP202 and E. coli S17-1, respectively. We also thank Dr. Makoto Hidaka (Tokyo University) for technical advice in the construction of A. brasiliense disruptant. We are grateful to Dr. Yasuhiro Takada and Tomohiro Hirose (Hokkaido University) for their help in the determination of amino acid sequences.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Zaldivar, J., Nielsen, J., and Olsson, L. (2001) Appl. Microbiol. Biotechnol. 56, 17-34[CrossRef][Medline] [Order article via Infotrieve]
  2. Stryer, L. (1988) Biochemistry, 3rd Ed., pp. 805-807, W. H. Freeman & Co., New York
  3. Richard, P., Londesborough, J., Putkonen, M., Kalkkinen, N., and Penttila, M. (2001) J. Biol. Chem. 276, 40631-40637[Abstract/Free Full Text]
  4. Richard, P., Putkonen, M., Vaananen, R., Londesborough, J., and Penttila, M. (2002) Biochemistry 41, 6432-6437[CrossRef][Medline] [Order article via Infotrieve]
  5. Verho, R., Putkonen, M., Londesborough, J., Penttila, M., and Richard, P. (2004) J. Biol. Chem. 279, 14746-14751[Abstract/Free Full Text]
  6. Welmberg, R., and Doudoroff, M. (1955) J. Biol. Chem. 217, 607-624[Free Full Text]
  7. Welmberg, R. (1961) J. Biol. Chem. 236, 629-635[Free Full Text]
  8. Dagley, S., and Trudgill, P. W. (1965) Biochem. J. 95, 48-58[Medline] [Order article via Infotrieve]
  9. Dahms, A. S., and Anderson, R. L. (1969) Biochem. Biophys. Res. Commun. 36, 809-814[CrossRef][Medline] [Order article via Infotrieve]
  10. Pedrosa, F. O., and Zancan, G. T. (1974) J. Bacteriol. 119, 336-338[Abstract/Free Full Text]
  11. Duncan, M. J. (1979) J. Gen. Microbiol. 113, 177-179
  12. Duncan, M. J., and Fraenkel, D. G. (1979) J. Bacteriol. 137, 415-419[Abstract/Free Full Text]
  13. Novick, N. J., and Tyler, M. E. (1982) J. Bacteriol. 149, 364-367[Abstract/Free Full Text]
  14. Mathias, A. L., Rigo, L. U., Funayama, S., and Pedrosa, F. O. (1989) J. Bacteriol. 171, 5206-5209[Abstract/Free Full Text]
  15. Kim, S. T., Huh, W. K., Lee, B. H., and Kang, S. O. (1998) Biochim. Biophys. Acta 1429, 29-39[CrossRef][Medline] [Order article via Infotrieve]
  16. Edwards, K. J., Barton, J. D., Rossjohn, J., Thorn, J. M., Taylor, G. L., and Ollis, D. L. (1996) Arch. Biochem. Biophys. 328, 173-183[CrossRef][Medline] [Order article via Infotrieve]
  17. Lamble, H. J., Heyer, N. I., Bull, S. D., Hough, D. W., and Danson, M. J. (2003) J. Biol. Chem. 278, 34066-34072[Abstract/Free Full Text]
  18. Zachariou, M., and Scopes, R. K. (1986) J. Bacteriol. 167, 863-869[Abstract/Free Full Text]
  19. Wiegert, T., Sahm, H., and Sprenger, G. A. (1997) J. Biol. Chem. 272, 13126-13133[Abstract/Free Full Text]
  20. Nurizzo, D., Halbig, D., Sprenger, G. A., and Baker, E. N. (2001) Biochemistry 40, 13857-13867[CrossRef][Medline] [Order article via Infotrieve]
  21. Johnsen, U., and Schonheit, P. (2004) J. Bacteriol. 186, 6198-6207[Abstract/Free Full Text]
  22. Sperka, S., Zehelein, E., Fiedler, S., Fischer, S., Sommer, R., and Buckel, P. (1989) Nucleic Acids Res. 17, 5402[Free Full Text]
  23. Bonete, M. J., Pire, C., Llorca, F. I., and Camacho, M. L. (1996) FEBS Lett. 383, 227-229[CrossRef][Medline] [Order article via Infotrieve]
  24. Selig, M., Xavier, K. B., Santos, H., and Schonheit, P. (1997) Arch. Microbiol. 167, 217-232[Medline] [Order article via Infotrieve]
  25. Angelov, A., Futterer, O., Valerius, O., Braus, G. H., and Liebl, W. (2005) FEBS J. 272, 1054-1062[Medline] [Order article via Infotrieve]
  26. Elzainy, T. A., Hassan, M. M., and Allam, A. M. (1973) J. Bacteriol. 114, 457-459[Abstract/Free Full Text]
  27. Lamble, H. J., Milburn, C. C., Taylor, G. L., Hough, D. W., and Danson, M. J. (2004) FEBS Lett. 576, 133-136[CrossRef][Medline] [Order article via Infotrieve]
  28. Theodossis, A., Walden, H., Westwick, E. J., Connaris, H., Lamble, H. J., Hough, D. W., Danson, M. J., and Taylor, G. L. (2004) J. Biol. Chem. 279, 43886-43892[Abstract/Free Full Text]
  29. Laemmli, U. K. (1970) Nature 227, 680-685[CrossRef][Medline] [Order article via Infotrieve]
  30. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
  31. Gennady, P. M. (2002) Handbook of Detection of Enzymes on Electrophoretic Gels, 2nd Ed., pp. 27-33, CRC Press, Inc., Boca Raton, FL
  32. Moore, S., and Link, K. P. (1940) J. Biol. Chem. 133, 293-311[Free Full Text]
  33. Gross, E. (1967) Methods Enzymol. 11, 238-255[CrossRef]
  34. Sambrook, J., Fritsch, E. F., and Maniatis, T. (2001) Molecular Cloning: A Laboratory Manual, 3rd Ed., pp. 6.33-7.45, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  35. Penning, T. M., and Jez, J. M. (2001) Chem. Rev. 101, 3027-3046[CrossRef][Medline] [Order article via Infotrieve]
  36. Simon, R., Priefer, U., and Puhler, A. (1983) Bio/Technology 1, 789-791
  37. Tarrand, J. J., Krieg, N. R., and Döbereiner, J. (1978) Can. J. Microbiol. 24, 967-980[Medline] [Order article via Infotrieve]
  38. Arimitsu, E., Aoki, S., Ishikura, S., Nakanishi, K., Matsuura, K., and Hara, A. (1999) Biochem. J. 342, 721-728
  39. Galbraith, M. P., Feng, S. F., Borneman, J., Triplett, E. W., de Bruijn, F. J., and Rossbach, S. (1998) Microbiology 144, 2915-2924[Abstract/Free Full Text]
  40. Wengenmayer, F., Ueberschär, K. H., Kurz, G., and Sund, H. (1973) Eur. J. Biochem. 40, 49-61[Medline] [Order article via Infotrieve]
  41. Blachnitzky, E. O., Wengenmayer, F., and Kurz, G. (1974) Eur. J. Biochem. 47, 235-250[Medline] [Order article via Infotrieve]
  42. Elshafei, A. M., and Abdel-Fatah, O. M. (2001) Enzyme Microb. Technol. 29, 76-83[CrossRef][Medline] [Order article via Infotrieve]
  43. Asada, Y., Aoki, S., Ishikura, S. Usami, N., and Hara, A. (2000) Biochem. Biophys. Res. Commun. 278, 333-337[CrossRef][Medline] [Order article via Infotrieve]
  44. Aoki, S., Ishikura, S., Asada, Usami, N., and Hara, A. (2001) Chem. Biol. Interact. 130, 775-784
  45. Wierenga, R. K., De Maeyer, M. C. H., and Hol, W. G. J. (1985) Biochemistry 24, 1346-1357[CrossRef]
  46. Rossmann, M. G., Liljas, A., Brändén, C.-I., and Banaszak, L. J. (1975) in The Enzymes (Boyer, P. D., ed) 3rd Ed., Vol. 11, pp. 61-102, Academic Press, New York
  47. Lott, J. S., Halbig, D., Baker, H. M., Hardman, M. J., Sprenger, G. A., and Baker, E. N. (2000) J. Mol. Biol. 304, 575-584[CrossRef][Medline] [Order article via Infotrieve]
  48. Cosgrove, M. S., Naylor, C., Paludan, S., Adams, M. J., and Levy, H. R. (1998) Biochemistry 37, 2759-2767[CrossRef][Medline] [Order article via Infotrieve]
  49. Cosgrove, M. S., Gover, S., Naylor, C. E., Vandeputte-Rutten, L., Adams, M. J., and Levy, H. R. (2000) Biochemistry 39, 15002-15011[CrossRef][Medline] [Order article via Infotrieve]
  50. Moore, R. A., Reckseidler-Zenteno, S., Kim, H., Nierman, W., Yu, Y., Tuanyok, A., Warawa, J., DeShazer, D., and Woods, D. E. (2004) Infect. Immun. 72, 4172-4187[Abstract/Free Full Text]
  51. Van Bastelaere, E., Lambrecht, M., Vermeiren, H., Van Dommelen, A., Keijers, V., Proost, P., and Vanderleyden, J. (1999) Mol. Microbiol. 32, 703-714[CrossRef][Medline] [Order article via Infotrieve]
  52. Kemner, J. M., Liang, X., and Nester, E. W. (1997) J. Bacteriol. 179, 2452-2458[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
S. Watanabe, M. Saimura, and K. Makino
Eukaryotic and Bacterial Gene Clusters Related to an Alternative Pathway of Nonphosphorylated L-Rhamnose Metabolism
J. Biol. Chem., July 18, 2008; 283(29): 20372 - 20382.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Watanabe, M. Yamada, I. Ohtsu, and K. Makino
{alpha}-Ketoglutaric Semialdehyde Dehydrogenase Isozymes Involved in Metabolic Pathways of D-Glucarate, D-Galactarate, and Hydroxy-L-proline: MOLECULAR AND METABOLIC CONVERGENT EVOLUTION
J. Biol. Chem., March 2, 2007; 282(9): 6685 - 6695.
[Abstract] [Full Text] [PDF]

Home page
 MicrobiologyHome page
N. J. Poysti, E. D. M. Loewen, Z. Wang, and I. J. Oresnik
Sinorhizobium meliloti pSymB carries genes necessary for arabinose transport and catabolism
Microbiology, March 1, 2007; 153(3): 727 - 736.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
C. Stephens, B. Christen, T. Fuchs, V. Sundaram, K. Watanabe, and U. Jenal
Genetic Analysis of a Novel Pathway for D-Xylose Metabolism in Caulobacter crescentus
J. Bacteriol., March 1, 2007; 189(5): 2181 - 2185.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Watanabe, N. Shimada, K. Tajima, T. Kodaki, and K. Makino
Identification and Characterization of L-Arabonate Dehydratase, L-2-Keto-3-deoxyarabonate Dehydratase, and L-Arabinolactonase Involved in an Alternative Pathway of L-Arabinose Metabolism: NOVEL EVOLUTIONARY INSIGHT INTO SUGAR METABOLISM
J. Biol. Chem., November 3, 2006; 281(44): 33521 - 33536.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Watanabe, T. Kodaki, and K. Makino
A Novel {alpha}-Ketoglutaric Semialdehyde Dehydrogenase: EVOLUTIONARY INSIGHT INTO AN ALTERNATIVE PATHWAY OF BACTERIAL L-ARABINOSE METABOLISM
J. Biol. Chem., September 29, 2006; 281(39): 28876 - 28888.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. J. J. Brouns, J. Walther, A. P. L. Snijders, H. J. G. van de Werken, H. L. D. M. Willemen, P. Worm, M. G. J. de Vos, A. Andersson, M. Lundgren, H. F. M. Mazon, et al.
Identification of the Missing Links in Prokaryotic Pentose Oxidation Pathways: EVIDENCE FOR ENZYME RECRUITMENT
J. Biol. Chem., September 15, 2006; 281(37): 27378 - 27388.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An addition or correction has been published
Right arrow All Versions of this Article:
281/5/2612    most recent
M506477200v2
M506477200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Watanabe, S.
Right arrow Articles by Makino, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Watanabe, S.
Right arrow Articles by Makino, K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement