Cloning, expression, and characterization of a Baeyer–Villiger monooxygenase from Pseudomonas fluorescens DSM 50106 in E. coli

Cloning, Expression and Characterization of Bacterial L-Arabinose 1-Dehydrogenase Involved

in an Alternative Pathway of L-Arabinose Metabolism*

Seiya Watanabe‡§¶||, Tsutomu Kodaki§||, Keisuke Makino§¶||**

From ‡ Faculty of Engineering, Kyoto University, Kyotodaigakukatsura, Saikyo-ku, Kyoto

615-8530, Japan, § Institute of Advanced Energy, Kyoto University, Gokasyo, Uji, Kyoto 611-0011,

Japan, ¶ International Innovation Center, Kyoto University, Yoshidahonmachi, Sakyo-ku, Kyoto

606-8501, Japan and || CREST, JST (Japan Science and Technology Agency), Gokasyo, Uji, Kyoto

611-0011, Japan

Running title: A Novel Bacterial L-Arabinose 1-Dehydrogenase

** Corresponding author: Institute of Advanced Energy, Kyoto University, Gokasyo, Uji, Kyoto

611-0011 Japan, Tel: +81-774-38-3517; Fax: +81-774-38-3524; E-mail: [email protected]

Azospirillum brasiliense converts L-arabinose

to -ketoglutarate via five hypothetical

enzymatic steps. We purified and

characterized L-arabinose 1-dehydrogenase

(EC 1.1.1.46), catalyzing the conversion of

L-arabinose to L-arabino- -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

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 indicate 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.

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http://www.jbc.org/cgi/doi/10.1074/jbc.M506477200The latest version is at JBC Papers in Press. Published on December 2, 2005 as Manuscript M506477200

Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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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

extensively investigated. Many bacteria

including Escherichia coli depend on protein

products of the araBAD operon, which contains

araB (ribulokinase, EC 2.7.1.16), araA

(L-arabinose isomerase, EC 5.3.1.4) and araD

(L-ribulose-phosphate 4-epimerase, EC 5.1.3.4)

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) produces L-arabinitol, L-arabinitol

4-dehydrogenase (EC 1.1.1.12) produces

L-xylulose, L-xylulose reductase (EC 1.1.1.10)

produces D-xylitol and D-xylulose reductase

(EC 1.1.1.9) produces D-xylulose. D-Xylulose is

then phosphorylated by xylulokinase (EC

2.7.1.17) 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- -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)1 and -ketoglutaric semialdehyde. The

last step is the NAD(P)+-dependent

dehydrogenation of the semialdehyde to

-ketoglutaric acid. The second pathway has the

same initial three steps, but L-KDA is cleaved

through an aldolase reaction to glycolaldehyde

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) is involved in the biosynthesis of

D-erythroascorbic acid (15). Glucose

1-dehydrogenase (GDH) is classified into two

types, pyrroloquinoline-quinone-dependent

GDH (EC 1.1.5.2) and nicotinamide adenine

dinucleotide (phosphate)-dependent GDH.

According to primary structure analysis, these

GDH types are not related. Nicotinamide

adenine dinucleotide (phosphate)-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

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1.1.99.28), 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(179))

and D-galactose 1-dehydrogenase (EC

1.1.1.48(120)) (21, 22). GDH (EC 1.1.118(119))

is involved in the non-phosphorylative

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- -lactone. It is

interesting that enzymes of the

non-phosphorylative 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

non-phosphorylative ED pathway (Figs. 1C, D).

In this study, we focused on a proposed

NAD(P)+-dependent dehydrogenase that

converts L-arabinose to L-arabino- -lactone

(referred to as L-arabinose 1-dehydrogenase, EC

1.1.1.46) (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

Bacterial Strain, Culture Conditions and

Preparation of Cell-free Extracts—Azospirillum

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 KH2PO4, 6.0 g

K2HPO4, 0.2 g MgSO4·H2O, 0.1 g NaCl, 0.026 g

CaSO4·2H2O, 1.0 g NH4Cl, 0.01 g FeCl3·6H2O,

0.002 g NaMoO4·2H2O, and 0.0001 g biotin per

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

× 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 use.

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

Incorporated, NY, USA) and then centrifuged at

108,000 × g for 20 min at 4oC to obtain cell-free

extracts.

Polyacrylamide Gel Electrophoresis

(PAGE)—SDS-PAGE was performed as

described by Laemmli (29). Non-denaturing

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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 R250 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.

Purification of L-Arabinose

1-Dehydrogenase—All purification steps were

performed below 4 °C. The cell-free extracts

were fractionated between 50–60% 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 ÄKTA

purifier system (Amersham Pharmacis Biotech).

After insoluble materials were removed by

centrifugation, the supernatant was applied to a

HiPrep 16/10 Butyl FF column (1.6×10 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

against a large volume of Buffer A for overnight.

The enzyme solution was loaded onto a column

of HiPrep 16/10 Q FF (1.6×10 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 CHT Ceramic

Zymogram Staining Analysis—The cell-free

extracts or purified enzyme were separated on

non-denaturing 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

nitroblue 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.

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Hydroxyapatite Type I (1.6 × 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 ×

g for approximately 2 h. The enzyme solution

was loaded onto a column of HiLoad 16/60

Superdex 200 pg (1.6×60 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 use.

The native molecular mass (Mr) 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. HMW and LMW 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

Multi-Station LC-8020 model II system

(TOSOH). L-Arabino- -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

(300×7.8 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 a Hybond™-P

(Amersham Biosciences) at 3 mA/cm2 for 0.5 h

in a transfer buffer (10 mM

cyclohexylaminopropane sulfonic acid, pH 11,

containing 10% (v/v) methanol) with a

horizontal electrophoretical 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

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a Procise™ 492 HT protein sequencer (Applied

Biosystems).

Chemical digestion with cyanogen bromide

(BrCN) 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) BrCN (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)

bromophenolblue 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

Hybond™-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/YA

A]CAT-3’. A. brasiliense genomic DNA was

prepared using a DNeasy™ Tissue Kit (Qiagen).

PCR was carried out using a PCR Thermal

Cycler PERSONAL (TaKaRa) for 30 cycles in

50 μl reaction mixture containing 10 pmol

primers, 1.25 U 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 CyDye™

Terminator Sequencing Kit (Veritas) and

appropriate primers with Long-Read Tower,

UBC DNA sequencer (Amersham Biosciences).

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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, approximately

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

Hybond™-N (Amersham Biosciences) by

capillary transfer using 10 × SSC as a transfer

buffer (1 × SSC is 15 mM sodium citrate, pH 7.0,

and 0.15 M NaCl). The blotted filter was

cross-linked in an UV Crosslinker 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 Molecular Biochemicals).

Membrane was visualized using a NBT/BCIP

reagent detection system (Roche Molecular

Biochemicals).

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

Molecular Biochemicals). 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

(OD600 = 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 (pH 7.0–7.2, 10 g

peptone, 10 g meat extract and 5.0 g NaCl) and

harvested by centrifugation. Total RNAs from A.

brasiliense were prepared with an RNeasy®

Mini Kit (Qiagen), and subsequently treated

with RNase-free DNaseI. 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 (small letters indicate

additional bases for introducing digestion sites

of BglII and PstI (underlined letters)):

5’-caccatagaTCTGATCAGGTTTCGCTGGGTG-

3’ (HISBglII) and

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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 (His)6-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: I) HISBglII

and one of the antisense primers containing the

mutations; and 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

(His)6-tagged L-Arabinose 1-Dehydrogenase—E.

coli DH5 harboring the expression plasmid for

the (His)6-tagged wild-type and mutated

enzymes was grown at 37 °C to a turbidity of

0.6 at 600 nm in Super broth medium (pH 7.0,

12 g tryptone, 24 g yeast extract, 5 ml glycerol,

3.81 g KH2PO4 and 12.5 g K2HPO4 per liter)

containing 50 mg/liter ampicillin. After the

addition of 1 mM of

isopropyl- -D-thiogalactopyranoside, the culture

was further grown for 6 h to induce the

expression of (His)6-tagged L-arabinose

1-dehydrogenase protein. Cells were harvested

and resuspended in Buffer D (pH 8.0, 50 mM

sodium phosphate containing 2 mM MgCl2, 300

mM NaCl, 1 mM L-arabinose, 10 mM

2-mercaptoethanol and 10 mM imidazole). The

cells were then disrupted by sonication, and the

solution was centrifuged. The supernatant was

loaded onto a Ni-NTA spin column (Qiagen)

equilibrated with Buffer D. The column was

washed 3 times with Buffer E (pH 8.0, Buffer D

containing 10% (v/v) glycerol and 50 mM

imidazole instead of 10 mM imidazole). The

enzymes were then eluted with Buffer F (pH 8.0,

Buffer E containing 250 mM imidazole instead

of 50 mM imidazole). The elutant was dialyzed

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against Buffer C and stored at –35 °C until use.

Western Blot Analysis of (His)6-tagged

L-Arabinose 1-Dehydrogenase—For Western

blot analysis, the purified L-arabinose

1-dehydrogenase from A. brasiliense and/or

recombinant (His)6-tagged L-arabinose

1-dehydrogenase from E. coli were separated by

SDS-PAGE, and the proteins on the gels were

transferred onto a nitrocellulose membrane

(Hybond™-ECL; Amersham Biosciences).

Western blot analysis was carried out using the

ECL™ Western Blotting Analysis System

(Amersham Biosciences) and RGS·His HRP

antibody, a horseradish peroxidase-fused mouse

monoclonal antibody against Arg-Gly-Ser-(His)6

in the N-terminal additional peptide of the

expressed recombinant proteins (Qiagen).

Disruptant Construction—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 (small letters indicate

additional bases for introducing digestion sites

of MfeI (underlined letters)):

5’-caccatcaattgGATCAGGTTTCGCTGGGTGT

CGTCGGCATCG-3’ (MfeI-up) and

5’-gcttggcaattgTCAGCGGCCGAACGCGTCG

GTCTGCACGCGC-3’ (MfeI-down). The

2.3-kbp MfeI DNA fragment was subcloned into

EcoRI site in 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

per 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

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 Clustal W program

distributed by DDBJ (DNA Data Bank of Japan)

(www.ddbj.nig.ac.jp). The phylogenetic tree was

produced using the TreeView 1.6.1. program.

RESULTS

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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 I. During

the purification procedure, the ratio of NADP+-

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 (Figs. 2A, C) and showed NAD+-

and NADP+-dependent specific activity of 25

and 44 units/mg protein, respectively (Table I).

SDS-PAGE revealed only one subunit with an

apparent Mr value of 39.5 ± 0.7 kDa (Fig. 2A).

To estimate native Mr, the enzyme was loaded

onto a HiLoad 16/60 Superdex 200 pg column.

The Mr value from the calibration curve with

marker proteins was approximately 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 dehydrogenase.

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 II. 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+ due to 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 (EC

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1.1.1.48(120))”. 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 two orders of magnitude in the

presence of either NAD+ or NADP+ due to

remarkably high Km values and low kcat values.

Enzyme activities with these sugars and the

stereo-configuration of these sugars are shown

in Fig. 3. Active sugars for L-arabinose

1-dehydrogenase belong to not only pentose(s)

but also hexose(s), indicating that the activity is

not dependent on the C5 and C6 configuration.

D-Galactose has the same configuration as

L-arabinose at C2, C3 and C4. The activity was

also found with D-talose, C1 epimer of

D-galactose but not with L-mannose and

D-glucose, C3 and C4 epimer, respectively.

Similarly, the activity with D-xylose, the C4

epimer of L-arabinose, decreased significantly

and no activity was observed with L-lyxose, the

C3 epimer of L-arabinose. It therefore appeared

that the enzyme prefers the L-arabinose-specific

configuration at C3 and C4.

It has been reported that the product of the

enzyme reaction in the cell-free extract system

is L-arabino- -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- -lactone was

slightly earlier than that of L-arabinose, and the

main product from L-arabinose was

L-arabino- -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- -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 BrCN,

which digests specifically at the C-terminals of

methyonyl residues in polypeptides. As shown

in Fig. 2B, two peptide bands with

approximately 26.4 and 0.92 kDa of molecular

weight (referred to as Fragments I and II,

respectively) were found on SDS-PAGE 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, a ~300-bp nucleotide

fragment of the L-arabinose 1-dehydrogenase

gene was obtained by genomic PCR. Southern

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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 approximately 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 to 13 bases upstream of the ATG codon.

The deduced amino acid sequence of the protein

(polypeptide of 309 amino acids with a

predicted Mr 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 BrCN was

found at positions 89 to 103 of the protein

sequences. The nucleotide sequence was

submitted to GenBank™ with accession number

AB211983. 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.

L-Arabinose 1-dehydrogenase was

overexpressed in E. coli cells by induction with

isopropyl- -D-thiogalactopyranoside as a

(His)6-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 (Figs. 6A, C) was

slightly later than that of the native enzyme

purified from A. brasiliense cells because of the

additional 13 amino acid residues including

(His)6-tag at the N-terminal. Western blot

analysis with anti-(His)6-tag antibody confirmed

the (His)6-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 II and III),

confirming that the isolated gene encodes

L-arabinose 1-dehydrogenase.

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),

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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), while L-arabinose

1-dehydrogenase possesses no lactonase activity

with L-arabino- -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

(subfamily I in Fig. 7B).

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.

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 due to

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 (Figs. 7A, 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 in “Experimental

procedures”, to construct D168A and N172A

mutants, respectively. They were overexpressed

in E. coli cells as a (His)6-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 four orders of

magnitude in kcat/Km values, compared with the

wild-type enzyme (Table III). 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

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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 II). 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: 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 (Figs. 8B, C) were analogous to those

observed at the level of transcription.

Furthermore, we constructed a A.

brasiliense disruptant of the L-arabinose

1-dehydrogenase gene, designated ARA5034,

by biparental mating and double homologous

recombination as described in “Experimental

procedures” (Fig. 9A). When 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 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.

DISCUSSION

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 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

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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

due to 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+; the S116D mutant

of ZmGFOR shows significant activity with

NAD+, 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 Ps. 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,

upward 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 C1 OH group of G6P,

and NAD(P)+ abstracts a hydride ion (H-) from

the C1 atom. Asp235 forms a hydrogen bond with

C2 OH of G6P. In ZmGFOR, the structurally

homologous Asp265–Tyr269 residues play

equivalent roles (Figs. 7A, 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 (Figs. 7A, 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 four-orders of magnitude (48) and a

10-fold higher Km value for G6P. Therefore, we

constructed two mutants of L-arabinose

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Physiological Insight of L-Arabinose

Metabolism in A. brasiliense—We could find

many L-arabinose 1-dehydrogenase gene

homologs on the bacterial genome sequence

(subfamily I in Fig. 7B). 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 (Fig. 5), which was isolated from a

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.

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 III 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 III), while 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 C1 OH of G6P 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 (Figs. 7B, C).

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

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

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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, 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. This

enzymatic and genetic evidence indicates that

the L-arabinose 1-dehydrogenase gene is only

involved in the metabolic pathway of

L-arabinose but 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

(Fig. 5) are absolutely necessary for growth on

L-arabinose in Burkholderia thiailandensis, a

near relative bacteria of B. cepacia strain

R18149; deletion mutants of one of these two

genes showed a L-arabinose-negative phenotype

as well as 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.

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, while

residual uptake of these sugars is found in the

sbpA mutant. These findings suggest the

existence of multiple transport systems in A.

brasiliense. Since 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.

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44. Aoki, S., Ishikura, S., Asada, Y., Usami, N., and Hara, A. (2001) Chem. Biol. Interact. 130-132,

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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.

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(2000) Biochemistry 39, 15002-15011

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FOOTNOTES

* 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. Our thanks extend to Dr. Makoto Hidaka of Tokyo University, for his technical advice in

the construction of A. brasiliense disruptant. We are grateful to Dr. Yasuhiro Takada and Mr.

Tomohiro Hirose, Hokkaido University, for their help in the determination of amino acid sequences.

This work was supported by the Center of Excellence (COE) program for the "Establishment of COE

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. This work was also supported by CREST of the Japan Science and Technology Corporation.

The nucleotide sequences data reported in this paper will appear in the DDBJ, EMBL and Genbank™

nucleotide sequence database with accession number AB211983.

1 The abbreviations used are: L-KDA, L-2-keto-3-deoxyarabonate; GDH, glucose 1-dehydrogenase;

ED pathway, Entner-Doudoroff pathway; GFOR, glucose-fructose oxidoreductase; PAGE,

polyacrylamide gel electrophoresis; Mr, molecular mass; BrCN, cyanogen bromide; DD, dihydrodiol

dehydrogenase; ZmGFOR, GFOR from Zymomonas mobilis; G6PDH, Glucose 6-phosphate

dehydrogenase; LmG6PDH, G6PDH from Leuconostoc mesenteroides.

FIGURE LEGENDS

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

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bacteria (second pathway). D. Non-phosphorylative ED pathway.

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 BrCN-digestion. The purified enzyme (100 μg) was digested chemically

with BrCN, and the solution corresponding to 5 μg protein was applied. Two observed bands except

undigested polypeptide were referred to as Fragments I and II with molecular weights of 26.4 and

0.92 kDa, respectively. C. Non-denaturing PAGE (lane 1) and zymogram-staining analysis (lanes 2 to

5). The purified enzyme of 5 μg and cell-free extracts of 100 μg were applied. NAD+ and NADP+ in

lanes 2, 4 and lanes 3, 5, respectively, were used as a coenzyme.

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 II 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.

Figure 4. HPLC analysis of the reaction products. L-Arabinose (A), L-arabino- -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.

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. The NotI-NotI DNA

fragment of A. brasiliense genome DNA was cloned in this study.

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 to 4, (His)6-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-PAGE gel. B. Western

blot analysis. One μg of protein was electrophoresed. Western blot analysis was carried out using

anti-(His)6 antibody. C. Zymogram staining analysis. One μg of protein was applied. After

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electrophoresis, the gel was soaked into staining solution in the presence of 100 mM L-arabinose and 1

mM NADP+.

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 Ps. fluorescens and

GFOR from Z. mobilis. A fifty-two-length 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 of 4

(upward arrow) and functioning as a catalytic base (open circle) are indicated below the sequences.

GenBank™ accession numbers for protein sequences are in Fig. 7B. B. The phylogenetic relationships

among bacterial L-arabinose/D-galactose 1-dehydroegnases (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 GenBank™ 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 bold letters.

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 ± SD, n = 3.

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

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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 ARA5034 strain of A. brasiliense on D-glucose ( ),

L-arabinose ( ), D-galactose ( ) and D-xylose ( ) as a sole carbon source. Each sugar was

supplemented at the concentration of 37 mM in a minimal medium. For ARA5034, 25 μg of

kanamycin per liter was added. Cell growth was monitored by measuring absorbance at 600 nm.

23

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Table I. Summary of L-Arabinose 1-Dehydrogenase Purification from A. brasiliense.Total activity (units)

Step Total

protein(mg) NAD+ NADP+ NADP+

NAD+

Specific activity (units/mg protein)

Yield (%)

Purificationfold

Cell-free extract 1488 248 373 1.50 0.25 100 1.0(NH4)2SO4 fractionation 1147 171 287 1.68 0.25 77 1.0HiPrep 16/10 Butyl FF 186 109 202 1.85 1.09 54 4.4HiPrep 16/10 Q FF 53 97 158 1.63 2.98 42 12CHT Ceramic Hydroxyapatite 7.8 70 124 1.77 15.9 33 64HiLoad 16/60 Superdex 200 pg 1st 4.2 51 91 1.78 21.7 24 87HiLoad 16/60 Superdex 200 pg 2nd 1.0 25 44 1.76 44.0 12 176

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Table II. Kinetic Parameters of L-Arabinose 1-Dehydrogenase.

Substrate Coenzyme Vmax(units/mg protein)

Km(mM)

kcat(min-1)

kcat/Km(min-1·mM-1)

L-arabinose a NAD+ 25.0±0.2 b 1.41±0.16 d 1140±100 d 806±17 d

NADP+

44.9±0.1 b 0.255±0.016 d 2000±40 d 7860±330 d

D-galactose a NAD+ 23.8±0.1 b 1.49±0.14 d 1190±90 d 798±16 d

NADP+ 35.6±0.1 b 0.109±0.003 e 1560±10 e 14400±400 e

D-talose a NAD+ 1.7±0.1 b 3.95±0.09 f 62.1±1.4 f 15.7±0.1 f

NADP+ 12.8±0.2 b 5.87±0.20 f 580±22 f 98.9±0.4 f

D-xylose a NAD+ 5.3±0.2 c 210±49 g 240±40 g 1.16±0.07 g

NADP+ 14.8±0.3 c 72.0±7.6 g 720±51 g 10.0±0.3 g

Stocked L-arabinose 1-dehydrogenase was dialyzed against 100 mM Tris-HCl, pH 9.0, containing 2 mM MgCl2, overnight at 4 oCbefore enzyme activity was measured. Values are the means ± SD, n = 3. a Enzyme activity was measured with 100 mM Tris-HCl, pH 9.0 containing 1 mM NAD(P)+.b Ten mM sugar was used as a substrate. c Five hundred mM D-xylose was used as a substrate. d Eleven different concentrations of sugar between 0.1 and 5.0 mM were used. e Ten different concentrations of D-galactose between 0.05 and 0.5 mM were used. f Five different concentrations of D-talose between 1 and 10 mM were used. g Ten different concentrations of D-xylose between 0.1 and 1.0 M were used.

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Table III. Kinetic Parameters of Recombinant L-Arabinose 1-Dehydrogenase.

Enzyme Coenzyme Vmaxa

(units/mg protein) Km

(mM)kcat

(min-1)kcat/Km

(min-1·mM-1)Wild-type NAD+ 14.9±0.1 0.785±0.048 b 366±15 b 467±10 b

NADP+

32.0±0.1 0.260±0.003 b 989±11 b 3810±10 b

D168A NAD+ <0.01 N.D. c N.D. N.D.NADP+ <0.01 N.D. N.D. N.D.

N172A NAD+ 0.042±0.001 42.5±2.7 d 7.00±0.36 d 0.164±0.002 d

NADP+ 0.148±0.001 28.9±3.5 d 19.7±1.9 d 0.69±0.02 d

Stocked L-arabinose 1-dehydrogenase was dialyzed against 100 mM Tris-HCl, pH 9.0, containing 2 mM MgCl2, overnight at 4 oC. Values are the means ± SD, n = 3. a Under standard assay conditions. b Ten different concentrations of L-arabinose between 0.1 and 1.0 mM were used. c No determination due to low activity. d Eleven different concentrations of L-arabinose between 1 and 100 mM were used.

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L-arabinose

L-arabino-γ-lactone

L-arabonate

L-2-keto-3-deoxyarabonate (L-KDA)

L-arabinose1-dehydrogenase(EC 1.1.1.46)

L-arabinonolactonase(EC 3.1.1.15)

L-arabonate dehydratase(EC 4.2.1.25)

pyruvate+

glycolaldehyde

L-KDA aldolase(EC 4.1.2.18)

A B

C

NAD(P)+

NAD(P)H

H2O

H2O

α-ketoglutaric semialdehyde

α-ketoglutarate

L-KDA dehydratase(EC 4.2.1.43)

α-ketoglutaric semialdehydedehydrogenase

(EC 1.2.1.26)

H2O

NAD(P)+

NAD(P)H

L-arabinose

L-arabinitol

L-xylulose

xylitol

D-xylulose

D-xylulose 5-phosphate

aldose reductase(EC 1.1.1.21)

L-arabinitol4-dehydrogenase(EC 1.1.1.12)

L-xylulose reductase(EC 1.1.1.10)

D-xylulose reductase(EC 1.1.1.9)

xylulokinase(EC 2.7.1.17)

NAD+

NADH

NAD(P)H

NAD(P)+

NAD+

NADH

ATP

ADP

NAD(P)H

NAD(P)+

L-ribulose

L-ribulose 5-phosphate

L-arabinose

L-arabinose isomerase(EC 5.3.1.4)

ribulokinase(EC 2.7.1.16)

D-xylulose 5-phosphate

L-ribulose-phosphate4-epimerase(EC 5.1.3.4)

ATP

ADP

D-glucose

D-glucono-δ-lactone

D-gluconate

glucose1-dehydrogenase(EC 1.1.1.118(119))

gluconolactonase(EC 3.1.1.17)

D-gluconate dehydratase(EC 4.2.1.39)

NAD(P)+

NAD(P)H

H2O

H2O

D-2-keto-3-deoxygluconate (D-KDG)

pyruvate+

glyceraldehyde

D

D-KDG aldolase

First pathway Second pathway

Figure 1 Watanabe et al.

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89,000

60,100

45,000

36,300

26,400

113,000

200,000

18,500

M 1 2 3 4 5 6 7 M60,10045,000

36,300

26,400

18,500

9,200

M

Fragment I

Fragment II

Undigested

A B

2 3 4

cell-free extractpurified protein

1

C

5

Figure 2 Watanabe et al.

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100

101

102

103

104

105

L-arabinose D-galactose D-talose D-xylose

CHO

OHH

HHO

HHO

OHH

CH2OH

CHO

HHO

HHO

HHO

OHH

CH2OH

CHO

OHH

HHO

OHH

CH2OH

CHO

OHH

HHO

HHO

CH2OH

C1

C2

C3

C4

C5

C6

CHO

OHH

OHH

HHO

HHO

CH2OH

L-mannose

CHO

OHH

HHO

OHH

OHH

CH2OH

D-glucose

CHO

OHH

OHH

HHO

CH2OH

L-lyxose

* * * * * *

kcat

/Km

(min

-1m

M-1

)F

isch

erpr

ojec

tion

Figure 3 Watanabe et al.

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0

100

200

300

400

0

100

200

300

400

0

100

200

300

400

0

100

200

300

400

mV

mV

mV

mV

5 5.5 6 6.5 7minutes

A

B

C

D

6.522

5.677

5.667

5.665

6.492

6.490

Figure 4 Watanabe et al.

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ORF1

ORF2

L-Arabinose1-dehydrogenase

NotI

NotI

A. brasiliense B. cepacia

Dihydrodipicolinate synthase/N-acetylneuraminate lyase(ZP_00218290)

Predicted dehydrogenase(ZP_00218291)

periplasmic component(ZP_00218292)

ATPase component(ZP_00218293)

permease component(ZP_00218294)

ABC-type sugar transport system

1 kbp

Dihydroxyacid dehydratase/phosphogluconate(ZP_00218289)

Transcriptional regulator(ZP_00218288)

Figure 5 Watanabe et al.

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A

B

C

60,100

45,000

36,300

26,400

18,500

1 2 3 4M

Figure 6 Watanabe et al.

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motif I

motif II

β1 α1 β2

α5β4 β5 α6

β8 β9 β10 β11

α8 α9

β6 α7 β7

A. brasiliense 1 ----------------------------SDQVSLGVVGIGKIARDQHLPAIDAEPGFKLTPs. fluorescens 1 ----------------------------MQPIRLGLVGYGKIAQDQHVPAINANPAFTLVZ. mobilis 53 ATLPAGASQVPTTPAGRPMPYAIRPMPEDRRFGYAIVGLGKYALNQILPGFAGCQHSRIE

A. brasiliense 33 ACASRHAEVTGVRNYRD-----------LRALLAAERELDAVSLCAPPQVRYAQARAALEPs. fluorescens 33 SVATQGKPCPGVENFQS-----------LGELLENGPPVDAIAFCTPPQGRFALVQQALAZ. mobilis 113 ALVSGNAEKAKIVAAEYGVDPRKIYDYSNFDKIAKDPKIDAVYIILPNSLHAEFAIRAFK

A. brasiliense 82 AGKHVMLEKPPGATLGEVAVLEALARERGLTLFATWHSRCASAVEPAREWLATRAIRAVQPs. fluorescens 82 AGKHVLVEKPPCATLGKAALWIKREQA---SAPCSPCIAYAPAIAAARDWLATRTLQSVQZ. mobilis 173 AGKHVMCEKPMATSVADCQRMIDAAKAANKKLMIGYRCHYDPMNRAAVKLIRENQLGKLG

A. brasiliense 142 VRWKEDVRRWHPGQ------QWIWEPGGLGVFDPGINALSIVTRILP-RELVLREATLIVPs. fluorescens 139 IDWKEDVRKWHPGQ------AWIWQPG-LGVFDPGINALSIVTHLLP-LPLFVESAELRVZ. mobilis 233 MVTTDNSDVMDQNDPAQQWRLRRELAGGGSLMDIGIYGLNGTRYLLGEEPIEVRAYTYSD

A. brasiliense 195 PSDVQTPIAAELDCADTDGVPVRAEFDWRHGP---VEQWEIAVDTADGVLAISRGGAQLSPs. fluorescens 191 PSNCQSPIAASIKMSDPRLLDVRAEFDFDHGH---DELWSIQIRCAEGTLRLDNGGALLSZ. mobilis 293 PNDERFVEVEDRIIWQMRFRSGALSHGASSYSTTTTSRFSVQGDKAVLLMDPATGYYQNL

A. brasiliense 252 IAGEP-----VELGP------EREYPALYAHFHALIAR--------GESDVDVRPLRLVAPs. fluorescens 248 IDGVR-----QTVAE------EGEYAAVYRHFQQLIGD--------KTSDVDVQPLRLVAZ. mobilis 353 ISVQTPGHANQSMMPQFIMPANNQFSAQLDHLAEAVINNKPVRSPGEEGMQDVRLIQAIY

A. brasiliense 293 DAFLFGRRVQTDAFGR-----Ps. fluorescens 289 DSFFVGSRVSVEAFYD-----Z. mobilis 413 EAARTGRPVNTDWGYVRQGGY

α3α2 β3 α4

Y00459

Z93940

AF059313

Z99118

638

449

X78503

M76431

408

772

955

U18133

AB021928AB021929

AB021933AB021931

731

1000

334

1H6DK(ZmGFOR)

AAK23207799

AMM40130AAM35776

1000

719

B75475

AAW78223

313

906

441

AAK22951BAC48470

E97495CAC45736 BAB47899

672996

AB3554

547

AB211983 (A.brasiliense)ZP_00218291 (B. cepacia)

1000 NP_794517

S04853 (Ps. fluorescens)

1000

752

NP_636135NP_641149YP_202443

1000

999

319

642

AB021930

320783

Subfamily I

Subfamily II

Subfamily III

Subfamily IV

A

B

Figure 7 Watanabe et al.

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L-Arabinose 1-dehydrogenase (AB211983) GLGVFDPG-INALSIVTRILPR

D-Galactose 1-dehydrogenase (S04853) -LGVFDPG-INALSIVTHLLPL

GFOR (1H6DK) GGSLMDIG-IYGLNGTRYLLGE

D-Xylose 1-dehydrogenase (AAW78223) GCAVMDIG-IYPLNTSRFLLDA

Dihydrodiol dehydrogenase (AB021931) GGALLDLG-IYCVQFISMVFGG

myo-Inositol dehydrogenase (AF059313) GGIFRDMT-IHDFDMARFLLGE

G6PDH (1DPG) AGALLDMIQNHTMQIVGWLAME

168. 172.

265. 269.

235. 240.

176. 180.

164. 168.

206. 210.

170. 174.

C

Figure 7 (continued) Watanabe et al.

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BN

0

0.1

0.2

0.3

0.4

Spe

cifi

cac

tivi

ty(u

nits

/mg

prot

ein)

1 2 3 4 5

A

C

rRNA

Figure 8 Watanabe et al.

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B1

pHISWT

(5647 bp)

B

B

1

pUC4K(3914 bp) Kmr

ECmr

E/M

E/M

1

pSUPWT::Km

(10023 bp)

1

pHISWT::Km

(6911 bp)

B

B

Kmr

Tcr

Kmr

A

1 Tcr

Ampr

pSUP202(7830 bp)

Ampr

0

1

2

3

4

5

6

0 10 20 30 40

0

1

2

3

4

5

0 10 20 30 40

B

Wild-type ΔARA5034

Time (h) Time (h)

A60

0

A60

0

Figure 9 Watanabe et al.

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VOLUME 280 (2005) 10001–10010

Activation of mitogen-activated protein kinase kinase (MKK) 3 and MKK6 by type I interferons.Yongzhong Li, Sandeep Batra, Antonella Sassano, Beata Majchrzak, David E. Levy, Matthias Gaestel, Eleanor N. Fish, Roger J. Davis, and Leonidas C. Platanias

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Due to an inadvertent error, the wrong immunoblots were included in Fig. 8, E and F. Fig. 8 should appear as shown below. The figure legend andtext remain unchanged.

FIGURE 8

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 15, pp. 10651–10652, April 14, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

APRIL 14, 2006 • VOLUME 281 • NUMBER 15 JOURNAL OF BIOLOGICAL CHEMISTRY 10651

ADDITIONS AND CORRECTIONS This paper is available online at www.jbc.org

We suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the originalarticle. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carrynotice of these corrections as prominently as they carried the original abstracts.

VOLUME 280 (2005) PAGES 19594 –19599

Vertebrate nonmuscle myosin II isoforms rescue smallinterfering RNA-induced defects in COS-7 cellcytokinesis.Jianjun Bao, Siddhartha S. Jana, and Robert S. Adelstein

PAGES 19594 –19595:

Under “Experimental Procedures,” subheading “DNA Constructsand RNAOligonucleotides,” the last sentence that begins on page 19594is incorrect. It should read as follows: “NMHC II-B siRNA duplexes(directed at both the human, RefSeq accession number NM_005964,and monkey sequence, AAGGAUCGCUACUAUUCAGGA) andNMHC II-C siRNA (directed at the human C1-inserted sequence,UCCGUCAGCACCGUGUCUUAU (S. S. Jana, unpublished data))were chemically synthesized by Dharmacon, Inc. (Lafayette, CO) andQiagen (Valencia, CA), respectively.”

VOLUME 281 (2006) 2612–2623

Cloning, expression, and characterization of bacterialL-arabinose 1-dehydrogenase involved in an alternativepathway of L-arabinose metabolism.Seiya Watanabe, Tsutomu Kodaki, and Keisuke Makino

PAGE 2612:

Dr. Kodaki’s last name was misspelled. The correct spelling is shownabove.

Additions and Corrections

10652 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 15 • APRIL 14, 2006

Keisuke MakinoWatanabe Seiya, Kodaki Tsutomu and  L-Arabinose MetabolismInvolved in an Alternative Pathway ofof Bacterial L-Arabinose 1-Dehydrogenase Cloning, Expression and CharacterizationEnzyme Catalysis and Regulation:

published online December 2, 2005J. Biol. Chem. 

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