Puriˆcation, Molecular Cloning, and Characterization of Pyridoxine 4-Oxidase from Microbacterium Luteolum

Biosci. Biotechnol. Biochem., 66 (5), 1022–1031, 2002

Puriˆcation, Molecular Cloning, and Characterization of Pyridoxine 4-Oxidase from Microbacterium luteolum

Yasuo KANEDA, Kouhei OHNISHI1,andToshiharuYAGI†

From the Department of Bioresources Science, Faculty of Agriculture, and the 1Research Institute of Molecular Genetics, Kochi University, Monobe-Otsu 200, Nankoku, Kochi 783-8502, Japan

Received November 14, 2001; Accepted December 25, 2001

Pyridoxine 4-oxidase (EC 1.1.3.12, PN 4-oxidase), oxide. In pathway II, found in Pseudomonas IA and which catalyzes the oxidation of PN by oxygen or other Arthrobacter sp.2), PN is ˆrst oxidized to isopyridox- hydrogen acceptors to form pyridoxal and hydrogen al by PN 5-oxidase, and then degraded in 4 subse- peroxide or reduced forms of the acceptors, respec- quent steps to 2-hydroxymethyl succinic semialde- tively, was puriˆed for the ˆrst time to homogeneity hyde, acetic acid, ammonia, and carbon dioxide. from Microbacterium luteolum YK-1 (＝Aureobacteri- Although all except three enzymes in the two um luteolum YK-1).1 The puriˆed enzyme required pathways (PN 4-oxidase, PL 4-oxidase, and 3- FAD for its catalytic activity and stability. The enzyme hydroxy-2-methylpyridine-4,5-dicarboxylic acid 4- was a monomeric protein with the subunit molecular decarboxylase) have been puriˆed to homogeneity mass of 53,000±1,000 Da. PN was the only substrate as and characterized,2) yet neither their protein struc- the hydrogen donor. Oxygen, 2,6-dichloroindophenol, tures, nucleotide sequences of genes encoding them, and vitamin K3 were good substrates as the hydrogen nor the mechanisms of inheritance and control of acceptor. The gene ( pno) encoding PN 4-oxidase was these inducible sets of enzymes have been identiˆed. cloned. The gene encodes a protein of 507 amino acid Furthermore, it has not been shown whether the residues corresponding to the molecular mass of the genes are arranged in an operon or speciˆed by chro- subunit. PN 4-oxidase was expressed in Escherichia coli mosomal or plasmid DNA. and found to have the same properties as the native en- PN 4-oxidase partially puriˆed from Pseudomonas zyme from M. luteolum YK-1. Comparisons of primary MA-1 was an FAD-dependent enzyme and de- and secondary structures with other proteins showed hydrogenated PN to PL with oxygen or 2,6- that the enzyme belongs to the GMC oxidoreductase dichloroindophenol as the hydrogen acceptor.3) So family. M. luteolum YK-1 has four plasmids. The pno far the structure or reaction mechanism of the en- gene was found on a chromosomal DNA. Search for zyme has not been reported. genes similar in sequence in other organisms suggested Here, we report the puriˆcation and properties of that a nitrogen-ˆxing symbiotic bacterium, Mesorhizo- PN 4-oxidase from Microbacterium luteolum,which bium loti, which harbors two plasmids, has a PN degra- was isolated in our laboratory.4) The primary struc- dation pathway I in chromosomal DNA. ture of the enzyme was determined based on cloning and expression of its gene in Escherichia coli.The

Key words: vitamin B6; metabolic pathway; GMC gene encoding PN 4-oxidase was found on a chro- oxidoreductase; ‰avoprotein mosomal DNA.

Free forms (pyridoxine, PN, pyridoxal, PL, and Materials and Methods pyridoxamine, PM) of vitamin B6 are degraded through two diŠerent but related pathways by bacter- Materials. Horseradish peroxidase and NZ-amine ia that can use PN as a sole source of carbon and were obtained from Wako Pure Chemical Industries, nitrogen.1) In pathway I, found in Pseudomonas Osaka, Japan. Malate dehydrogenase and NADH MA-1, PN is ˆrst oxidized to PL by PN 4-oxidase, were obtained from Oriental Yeast, Tokyo, Japan. and then degraded through the following 7 steps to V-8 protease was from Sigma. Rat liver PL kinase, succinic semialdehyde, ammonia, and carbon di- and the apoform of E. coli aspartate aminotran-

† To whom correspondence should be addressed: Toshiharu YAGI, Phone and Fax: +81-88-864-5191 E-mail: yagito＠cc.kochi-u.ac.jp The nucleotide sequence reported in this paper has been submitted to the DDBJWGenBankWEBIDataBankwiththeaccessionnumber AB049341. 1 Aureobacterium luteolum was recently renamed Microbacterium luteolum [Takeuchi, M. and Hatano, K., Int. J. Syst. Bacteriol., 48, 739–747 (1998)]. Abbreviations: PN, pyridoxine; PL, pyridoxal; PMSF, phenylmethanesulfonyl ‰uoride; GMC, glucose-methanol-choline; AAT, aspar- tate aminotransferase Pyridoxine 4-Oxidase from Microbacterium luteolum 1023 sferase (AAT), were prepared as described previous- the suspension was sonicated again. The procedure ly.5) Ultrogel AcA 34 and Super Q-Toyopearl were was repeated 3 times. The combined cell extract from Separacor and Tosoh, respectively. Competent (165 ml) was used as the enzyme solution. Step 2: The cells of E. coli JM109, and a plasmid pTrc99A were crude extract was put on a DE52 column (3.8× from Takara and Amersham Pharmacia, respec- 24 cm) equilibrated with BuŠer A (20 mM potassium tively. All other chemicals were of analytical grade. phosphate buŠer, pH 8.0, containing stabilizing rea- gents: 5 mM FAD, 10z glycerol, 1z 2-mercap- Culture conditions. The bacterium was grown toethanol, and 0.01z Tween 20). The column was aerobically in the PN medium4) (5 ml in a test tube) at washed with BuŠer A until the absorption of the elu- 309C for 2-3 days. This broth was put into 100 ml of ate at 280 nm was lower than 0.1. The enzyme activi- the PN medium in a 500-ml shaking ‰ask and incu- ty was eluted with BuŠer A containing 0.15 M NaCl bated under the same conditions. The culture broth when the column was eluted with a linear gradient was centrifuged aseptically at 10,000×g for 10 min at (0–0.5 M NaCl). Step 3: The eluted enzyme solution 49C to collect the cells, which were then resuspended (580 ml) was dialyzed thoroughly against 5 mM ˆrst in a small amount and then in 500 ml of the PN potassium phosphate buŠer, pH 8.0, containing the medium in a 2-L shaking ‰ask, which was incubated stabilizing reagents. The dialyzed enzyme solution for 48 h before harvesting the cells. was put on a hydroxyapatite column (2.5×24 cm) equilibrated with the dialysis buŠer. The column was Enzyme and protein assays. The reaction mixture washed as described above. The enzyme activity was (0.4 ml) consisted of 0.1 M Tris-acetate buŠer (pH eluted with 25 mM potassium phosphate buŠer, pH 7.0), 5 mM PN, and enzyme, was incubated aerobi- 8.0, containing the stabilizing reagents when the cally at 309C for 30 min. The reaction was stopped column was eluted with a linear gradient elution by by addition of 66 mlof9M H2 SO4. The resulting solu- changing the concentration of the buŠer from 5 mM tion was centrifuged at 10,000×g for 5 min. The su- to 100 mM. Step 4: The eluted enzyme solution pernatant solution (0.3 ml) was mixed with 0.6 ml of (125 ml) was dialyzed thoroughly against BuŠer B

1 M H2 SO4 and 0.1 ml of 2z phenylhydrazine (50 mM Tris acetate buŠer, pH 7.0, containing the dissolved in 5 M H2SO4 and incubated at 609Cfor stabilizing reagents), and then put on a SuperQ- 20 min. The phenylhydrazone of PL formed was as- Toyopearl column (3.8×20 cm) equilibrated with sayed by measuring the absorbance at 410 nm.6) One BuŠer B. The enzyme activity was eluted with BuŠer unit of enzyme was deˆned as the amount that cata- B containing 0.05 M NaCl when the column was elut- lyzes the formation of 1 mmole of PL per min. ed with a linear gradient (0–0.5 M NaCl). Step 5: The The substrate speciˆcity was identiˆed with 5–10 eluted enzyme solution (60 ml) was dialyzed thor- fold higher amounts of the enzyme than those used in oughlyagainstBuŠerC(50mM ethanolamine buŠer, the standard assay by two other assay methods. The pH 9.0, containing the stabilizing reagents), and then assay based on oxygen consumption was done in a concentrated to 7 ml with a small SuperQ-Toyopearl reaction mixture (2.0 ml) consisting of 0.1 M potassi- column. The concentrated enzyme solution was put um phosphate buŠer (pH 8.0), the enzyme, 5.0 mM on an Ultrogel AcA 34 column (1.8×70 cm) PN, 5.0 mM FAD, and 0.01z Tween 20 with a oxyg- equilibrated with BuŠer A containing 0.1 M KCl. The raph equipped with a Clark electrode. The assay column was eluted with BuŠer A at the ‰ow rate of based on the measurement of H2 O2 produced was 0.2 mlWmin. The enzyme activity was eluted as a sin- done in a reaction mixture (1.0 ml) consisting of gle peak. Step 6: The eluted enzyme solution (10 ml) 0.1 M potassium phosphate buŠer (pH 8.0), the was dialyzed against BuŠer C, and then put on a enzyme, 5.0 mM PN, 10 mgperoxidase,1.0mM 4- SuperQ-Toyopearl column (1.8×8 cm) equilibrated aminoantipyrine, and 6.0 mM dichloro-2-hydroxy- with BuŠer C. The enzyme was eluted with BuŠer C benzensulfate.7) containing 0.1 M NaCl when the column was eluted Protein was measured by the dye-binding method8) with a linear gradient (0–0.2 M NaCl). with bovine serum albumin as a standard. Molecular mass measurement. The purity of the Puriˆcation of PN 4-oxidase. All steps were done enzyme and the subunit molecular mass were meas- at 4–109C. Step 1: The harvested cells (51.7 g) were ured by SDS-PAGE by the method of Laemmli.9) suspendedin55mlof20mM potassium phosphate Themolecularmassoftheenzymewasestimatedby buŠer (pH 8.0) containing 20 mM FAD, 1z 2-mer- gel ˆltration at 49C, using a Cosmosil 5 Diol 300 captoethanol, 10 z glycerol, 1 mM EDTA, and 1 mM HPLC column (0.75×60.0 cm) equilibrated with PMSF. The suspension was sonicated at 4–159Cfor BuŠer A containing 0.1 M KCl at a ‰ow rate of 20 min with a Heatsystems-Ultrasonics sonicator 1.0 mlWmin. A calibration curve was made from the W-220. The cell extract was obtained by centrifuga- elution pattern of bovine liver catalase (240,000 Da), tion at 10,000×g for 10 min at 49C. The precipitate pig heart mitochondrial aspartate aminotransferase was resuspended in the same volume of the buŠer; (90,000 Da), malate dehydrogenase (67,000 Da), and 1024 Y. KANEDA et al. horse heart cytochrome c (12,000 Da). pCR 2.1 vector supplied in a TA cloning kit (Invitro- gen). The constructed plasmid, pCRPNO1, was in- Amino acid sequencing. The enzyme (2.0 mg) was troduced into and extracted from E. coli TOP10F?. digested at 379Cfor2hwith0.4mg of V-8 protease The extracted pCRPNO1 was digested with Eco RI: in a reaction mixture consisting of 50 mM NH4HCO3, pCR 2.1 vector has Eco RI sites at both sides of the 2 M urea, and 0.2z SDS. The peptides were separat- cloning region. The Eco RI fragment having an en- edonaSDS-PAGEgel,andtransferredtoPVDF coding region for PN 4-oxidase was ligated into the membrane with a Bio-Rad semi-dry transfer appara- Eco RI site of pTrc 99A (Pharmacia). The construct- tus. The internal amino acid sequence was analyzed ed plasmid was designated as pTPNO1. with the main peptide on the membrane. The amino The PN 4-oxidase-encoding sequence in pTPNO1 terminal sequence of the enzyme was analyzed with was ampliˆed by PCR with the two oligonucleotides, undigested enzyme protein on the membrane. The se- 5?-CGGAATTCCGAGGAGGACGGTTAATGG- quencing was done by automated Edman degrada- CTCAGTATGATGTGGCGATC-3? (primer P6, tion with an Applied Biosystems 492 protein sequen- sense) and 5?-CCCAAGCTTGGGTTATTTGAAT- cer. CCTGTTGTGAAATT-3? (primer P7, antisense) to introduce Eco RI (underlined in primer P6), Analysis and cloning of the structural gene of PN ribosome-binding (boldface types in primer P6), and 4-oxidase. The structure of PN 4-oxidase-encoding Hind III sites (underlined in primer P7). The ampli- gene was analyzed as follows. Degenerate PCR ˆed DNA fragment (PNO2) was digested by Eco RI primers, P1 (sense) and P2 (antisense), were designed and Hind III and then ligated into the Eco RI-Hind based on the amino-terminal and internal sequences III site of pTrc 99A. The constructed plasmid was of the enzyme, respectively. The primers used were designated as pTPNO2. 5?ATGGC(A WG WT WC)CA(A WG)TA(T WC)GA(T W These plasmids, pTPNO1 and pTPNO2, were in- C)GT(AWGWTWC)GC(AWG WTWC)AT(AWT WC)AT(AW troduced into E. coli JM109-competent cells. The TWC)CG (primer P1) and 5?TA(AWGWTWC)GC(AW clone cells were used for analysis of the enzyme G)TT(A WG WT WC)CC(A WG WT WC)GG(A WG WT W production in E. coli. C)GC(A WG WT WC)GG(A WG WT WC)GC(A WG WT W C)GT(AWG)AA (primer P2). The chromosomal Puriˆcation of recombinant PN 4-oxidase. Cells of DNA of M. luteolum YK-1 prepared by the method the E. coli JM109 clone harboring pTPNO2 were of Saito and Miura10) wasusedasatemplate.The grown in 4 liters of LB Medium (2z,wWv, peptone, conditions for PCR, using the Takara LA Taq DNA 1z yeast extract, and 1z sodium chloride) contain- polymerase, were principally the same as described ing ampicillin (50 mgWml) and isopropyl-b-D-thio- previously.11) The nucleotide sequence of the ampli- galatopyranoside (1 mM)at309C for 12 h. The cells ˆed DNA fragment (1071 bp) was analyzed using a (wet weight of 13 g) were harvested and disrupted by PRISM DNA sequencing kit and an Applied Bio- sonication to prepare a crude extract. PN 4-oxidase systems 373A DNA sequencer. For sequencing of the (0.92 mg of total protein; 20.1 units of total activity) unknown DNA region encoding carboxyl side of the was puriˆed from the crude extract by essentially the enzyme, cassette-ligation mediated PCR was done same puriˆcation procedure as that used for puriˆca- withaTakaraLAPCRin vitro cloning kit. The cas- tion of the enzyme from M. luteolum YK-1. sette DNA containing a Eco RI site was ligated to the Eco RI-digested chromosomal DNA from M. luteo- Identiˆcation of the reaction product. HPLC lum YK-1, and the ligated fragments were used as the method: the enzyme reaction was done for 60 min in template DNA for PCR for the unknown 3? side the reaction mixture with 0.05 units of enzyme as region. The ˆrst PCR was done with a cassette primer described above. After the enzyme reaction was

C1 (antisense), and 5?-AGCCGGTGGCCAGCC- stopped by addition of H2 SO4, the reaction mixture GGAAATTGTCGTTGG-3?(primer 3, sense). The was centrifuged. The supernatant solution (10 ml) product of the ˆrst PCR was used as the template was diluted 1,000 times with distilled water, mixed DNA for the second PCR with a cassette primer C2 with 0.33 ml of 9 M perchloric acid, and then ˆltered and 5?-CGTGGCTCCAATCGTTTCCGAGAGCT- withaDISMIC13CPˆlter.Theˆltrate(100ml) was TTAC-3?(primer P4). The DNA fragment (about put through HPLC by the method described previ- 2.2 kb) encoding the 3? side region was ampliˆed. ously.12) ApoAAT method: after enzymatic reaction The partial DNA sequence (768 bp) of the fragment, (60 min), the reaction mixture was ultraˆltrated with in which a stop codon was found, was analyzed. a Centrifree. The ˆltrate (10 ml), was incubated at A DNA fragment (PNO1, 1587 bp) was ampliˆed 309Cfor60minin50mlof75mM potassium phos- by PCR with the chromosomal DNA as a template, phate buŠer (pH 6.0) containing PL kinase (9 units), the primer P1, and 5?-AACCGTCGTCAGCGTCA- 0.01 mM ZnSO4 and 0.1 mM ATP. Then the reaction GCGTTCCGCGTCCCAACTG-3?(primer P5) in a mixture (20 ml) was incubated with apoAAT (5 ng) in GC buŠer II (Takara). PNO1 was ligated into the 60 ml of Tris-HCl buŠer (pH 8.5) at 379Cinthedark. Pyridoxine 4-Oxidase from Microbacterium luteolum 1025

Table 1. Puriˆcation of Pyridoxine 4-Oxidase from M. luteolum

Total protein Total activity Speciˆc activity Yield Steps (mg) (units) unitsWmg (z) Crude extract 3380 157 0.05 100 DEAE-cellulose 979 119 0.12 75.8 Hydroxyapatite 53.8 54.5 1.01 34.7 Super Q-Toyopearl (pH 7.0) 5.45 47.6 8.73 30.3 Ultrogel AcA34 2.31 39.5 17.1 25.2 Super Q-Toyopearl (pH 9.0) 1.27 26.8 21.1 17.1

Fig. 1. SDS-PAGE of Crude and Puriˆed Preparations of Pyridoxine 4-Oxidase. Lane A, crude extract (55.2 mg of protein); lane B, a puriˆed fraction (33.6 mg) from the DE52 column; lane C, the fraction (8.7 mg) from the hydroxyapatite column; lane D, the fraction (1.9 mg) from the Super Q-Toyopearl column; lane E, the fraction (0.7 mg) from the Ultrogel AcA 34 column; and lane F, the fraction (0.6 mg) from the Super Q-Toyopearl column (pH 9.0). Molecular mass markers were rabbit muscle phosphorylase (97,400 Da), bovine serum albumin (66,200 Da), hen egg white ovalbumin (45,000 Da), bovine car- bonic anhydrase (31,000 Da), soybean trypsin inhibitor (21,500 Da), and hen egg white lysozyme (14,400 Da).

AAT activity was measured by the MDH-coupling lambda 50-kb DNA (New-England Bio Lab) were method described previously.5) used as size standards. The DNA bands were stained by ethidium bromide. Pulsed-ˆeld gel electrophoresis and Southern blot- A DNA fragment containing a gene encoding PN ting. The cells of M. luteolum YK-1 were grown 4-oxidase was detected with a ECL Direct Nucleic aerobically in LB medium (5 ml in a test tube) at Acid Labeling and Detection System (Amersham 309C to OD 600 of 0.8. Then chloramphenicol Pharmacia). The PNO1 fragment was used for label- (180 mgWml) was added and incubation was continued ing with peroxidase and the labeled PNO1 was used at 309C for 1 h. The cells were harvested by centrifu- as a probe. gation at 10,000×g and washed twice with 10 mM Tris-HCl (pH 7.6) containing 1 M NaCl. The cells Other procedures. Plasmids in M. luteolum YK-1 (2–5×107) were embedded in a gel plug of 1.0z were isolated with a NucleoBond BAC 100 kit (wWv) InCert agarose (FMC corporation) with a (Macherey-Nagel). Hydrogen peroxide was also CHEF genomic DNA plug kit (Bio-Rad). Plugged measured by the luminol-peroxidase method.13) samples were placed in slots of a running gel made of Photon counting was done with a Aloka BLR-201 0.8z (wWv) pulsed-ˆeld certiˆed agarose (Bio-Rad), luminescence reader. Activity staining of PN 4- andthenˆxedwithmolten1.0z Nusieve GTG oxidase was done by the method of Tsuge and agarose (FMC corporation) in the slots. Pulsed-ˆeld Nakanishi14) with some modiˆcation. The enzyme gel electrophoresis was done with a CHEF model solution (4 mg of protein) was put through PAGE by DR-II apparatus (Bio-Rad) under the following con- the method of Davis.15) The gel was transferred into ditions: electrophoresis buŠer, 0.5×Tris-Borate- 20 ml of an activity staining buŠer composed of EDTA;voltage,150V(4.5VWcm); reorientation 20 mM potassium phosphate buŠer (pH 8.0), 5 mM angle, 1209; pulse time, 60–120 sec; run time, 25 h; PN, 5 mM FAD, 0.25z o-dianisidine, and 5 mg temperature, 109C. Chromosomal DNA of Sac- horseradish peroxidase, and then incubated at 309C charomyces cerevisiae YNN295 and concatemers of for 30 min. Km and Vmax values were measured with 1026 Y. KANEDA et al.

toethanol, and 20 mM FAD before the Ultrogel AcA 34 column chromatography. However, after the step, the enzyme became unstable without addition of 0.01z Tween 20 to the buŠer. The puriˆed enzyme showed a single protein band with a molecular mass of 53,000±1,000 Da (average and S.D. of 3 experiments) on a SDS-PAGE gel (Fig. 1). The molecular mass of the native enzyme was 50,000±12,000 Da by gel ˆltration. The enzyme is thus a monomeric protein. The optimum pH measured in a reaction mixture containing 0.1 M sodium pyrophosphate buŠer (pH 6.0¿10.5) instead of 0.1 M Tris-acetate was 7.5–8.0; 48z and 5z of the maximal activity was displayed at pH 6.0 and pH 10.5, respectively. The optimum temperature was 409C; 30z of the maximal activity was displayed at 509C. The activa- tion energy calculated from an Arrhenius plot was 7,890±360 calWmol.

Identiˆcation of PL as an oxidation product of PN and stoichiometry of the oxidation reaction The reaction product was eluted at the same reten- tion time (13.00 min) as that of standard PL by the isocratic HPLC. When standard was added to the reaction mixture, it was coeluted with the reaction product. The reaction product phosphorylated by PL kinase activated apo-AAT, showing that the phos- phorylated product was PLP. Thus, the reaction

product is PL but not isopyridoxal. H2O2 and PL were produced in equimolar amounts.

Substrate and cofactor speciˆcity All substrates showed Michaelis-Menten kinetics in the concentration range studied. PN 4-oxidase showed reactivity only toward PN (Km, 54.5± 5.7 mM;kcat,28.1s„1 ) among the hydrogen donors Fig. 2. Nucleotide Sequence of Pyridoxine 4-Oxidase Gene of examined. Pyridoxamine, PL, 4-deoxypyridoxine, M. luteolum. YK-1 and deduced amino acid sequence of the enzyme. pyridoxal 5?-phosphate, pyridoxamine 5?-phosphate, The amino-terminal amino acid sequence and internal amino benzyl alcohol, 2-hydroxybenzyl alcohol, 3- acid sequence of V8 peptide indicated by underlines were ana- hydroxybenzyl alcohol, 2,3-dihydroxypyridine, lyzed by Edman degradation. 2,4-dihydroxypyridine, 2,6-dihydroxypyridine, 2- pyridinemethanol, 3-pyridinemethanol, and 4- pyridinemethanol were not substrates. In contrast to concentrations of substrate between 0.1×Km and this strict speciˆcity for the hydrogen donor, the en- 10×Km. Kinetic parameters were measured by using zyme showed a broad speciˆcity for hydrogen accep- curve-ˆtting software (kaleidaGraph) as described tors. O2 (Km, 206.64±18.8 mM), 2,6-dichloroin- 11) previously. dophenol (9.71±1.65 mM), and vitamin K3 (50.16± 14.6 mM) were good substrates. However, the enzyme Results could not use NAD+ or NADP+ as the substrate. PN 4-oxidase could use FAD (KD,0.16±0.01 mM) Puriˆcation and some properties of PN 4-oxidase as a cofactor, but not FMN. The enzyme was puriˆed to homogeneity from the crude extract of M. luteolum YK-1byˆvestepsof Amino acid and nucleotide sequences of PN column chromatography (Table 1). SDS-PAGE of 4-oxidase preparations from each steps are shown in Fig. 1. The amino acid sequences of the amino-terminal PN 4-oxidase was stable in a buŠer containing region and peptides from V8 protease digest of the stabilizing agents, 10z glycerol, 1z 2-mercap- puriˆed enzyme were analyzed (Fig. 2, underlines). Pyridoxine 4-Oxidase from Microbacterium luteolum 1027

Fig. 3. Expression of Pyridoxine 4-Oxidase in E. coli Transformed by Recombinant Plasmids.

A, Five colonies of E. coli JM 109WpTrc99A, JM109WpTPNO1, and JM109WpTPNO2 clones were isolated. They were individually grown in 100 ml of LB medium containing 50 mgWml ampicillin and 1 mM isopropyl-b-thiogalactopyranoside. PN 4-oxidase activity in the crude extracts of cells was measured. The activity was expressed as the speciˆc activity (unitWmg, protein) B, Representative pattern of SDS-PAGE. Lane Sd, molecular weight markers; lane A, B and C, crude extract of E. coli JM109WpTrc99A, JM109WpTPNO1, JM109WpTPNO2, respectively. ``PNO'' shows a protein band corresponding to that of PN 4-oxidase.

The amino terminus was alanine. The nucleotide Sequence comparisons sequence of the gene, identiˆed as described under The amino acid sequence of PN 4-oxidase was ``Materials and Methods'', encoded a 507 amino acid compared with other protein sequences by the protein (Fig. 2) with a predicted molecular mass BLAST program. A probable dehydrogenase protein (54,161 Da) in good agreement with the molecular of a nitrogen-ˆxing symbiotic bacterium, Mesorhizo- mass described above. bium loti,16) showed the highest similarity (Table 2). Choline dehydrogenase of E. coli17,18) and other GMC Cloning and expression of PN 4-oxidase gene oxidoreductases showed about 30z identity: The PN 4-oxidase gene ( pno) was ligated into representative enzymes, the protein structures of pTrc99A, and the recombinant plasmids (pTPNO1 which have been analyzed, were shown in order of and pTPNO2), in which pno wasinsertedasthesame theBitscoresintheTable2. direction as the promoter sequence, were used to Figure 4 shows the alignment of PN 4-oxidase with transform E. coli JM109 cells. The crude extracts of several enzymes including a probable dehydrogenase the transformed cells showed PN 4-oxidsase activity from M. loti, D.radiodurans GMC oxidoreductase, (Fig. 3A). Introduction of the ribosome-binding E. coli choline dehydrogenase, Pseudomonas sp. sequence increased expression of the enzyme 5-fold. TW3 4-nitrobenzyl alcohol dehydrogenase,19) and A. The crude extract from E. coli JM109WpTPNO2 niger glucose oxidase.20) The predicted secondary ele- showed a protein band corresponding to PN 4-oxi- ments of PN 4-oxidase and the secondary elements dase on a SDS-PAGE gel (Fig. 3B). found in glucose oxidase are shown. PN 4-oxidase shares common amino acid residues through its Properties of recombinant PN 4-oxidase whole sequence with the GMC oxidoreductases. Nine PN 4-oxidase from E. coli JM109WpTPNO2 secondary elements (A3, H1, A2, H6, A1, B2, B3, showed the same enzymatic properties such as the op- A5, and H13) out of 11 secondary elements, which timum pH, Km for PN, the molecular mass, speciˆc are involved in binding of FAD in the glucose oxi- activity, and amino-terminal 10 amino acids se- dase, were also found in the predicted secondary ele- quence, as the enzyme from M. luteolum YK-1: the ments in PN 4-oxidase. And 4 secondary elements amino-terminal amino acid residue was alanine. (H2, H8, H9 and C4) out of 9 secondary elements that are involved in the biding of substrate in the former enzyme were found in the latter enzyme. 1028 Y. KANEDA et al.

Table 2. Some Enzymes Similar to Pyridoxine 4-Oxidase from M. luteolum Accession indicates the accession numbers of sequence data (DDBJWEMBLWGenBank). Length is the number of amino acid residues of the enzyme proteins. Scores of Bit, Identity, and Similarity of the enzymes with PN 4-oxidase were obtained by the BLAST search in the DDBJ Homology Search Systems.

Source Enzyme Accession Length Bit Identity Similarity

Mesorhizobium loti Unknown protein* AP003010 520 688 66 78 E. coli Choline dehydrogenase M77738 556 233 33 48 Deionococcus radiodurans GMC oxidoreductase AE001949 529 233 33 47 Sphingomonas macrogolutabidus Polyethylene glycol AJ277295 533 229 31 48 dehydrogenase Pseudomonas sp. TW3 4-Nitrobenzyl alcohol AF043544 532 225 31 51 dehydrogenase Staphylococcus xylosus Choline dehydrogenase AF009415 560 222 34 47 Pseudomonas putida Alcohol dehydrogenase AJ245436 558 220 34 48 Pseudomonas oleovorans Alcohol dehydrogenase S27994 558 220 34 48 Pseudomonas aeruginosa Choline dehydrogenase AE004949 561 217 32 47 Staphylococcus aureus Choline dehydrogenase AP003137 569 203 32 45 Aspergillus niger Glucose oxidase A35459 605 130 28 41

* A mll6875 gene-encoding protein.

Fig. 4. Comparison of Pyridoxine 4-Oxidase with GMC Oxidoreductases. The amino acid sequences of Microbacterium luteolum PN 4-oxidase (MLPO), a probable dehydrogenase protein from Mesorhizobi- um loti (MLPD), Deionococcus radiodurans GMC oxidase (DRGO), E. coli choline dehydrogenase (ECCD), Pseudomonas sp. TW3 4- nitrobenzyl alcohol dehydrogenase (PPND), and Aspergillus niger glucose oxidase (ANGO) were aligned using CLUSTAL W (1.8). The shaded type shows an amino acid residue common in 5 or 6 sequences. Number of amino acid residue of ALPO, where methionine was numbered as the ˆrst residue, is shown above the sequence of ALPO. Amino acid residues comprising a-helix and b-sheet in ALPO, which were predicted as described by Ito et al.23), are shown by double and single lines under its sequence, respectively. The secondary structures found in ANGO, the tertiary structure of which has been determined,24) and their names are shown under the sequence of ANGO with single and double lines for b-sheet and a-helix, respectively. Pyridoxine 4-Oxidase from Microbacterium luteolum 1029

Fig. 5. Number of Plasmids and Localization of pno Gene. Pulsed-ˆeld electrophoreses were done for 25 h (A) and 6 h (B) under the conditions described under ``Materials and Methods''. A: lane 1, chromosomal DNA of Saccharomyces cerevisiae; lane 2, concatemers of lambda 50 kb DNA; lane 3, M. luteolum cells. A–D shows detected pno-containing band on the lane 3 by ECL Direct detection system. B: lane 1, chromosomal DNA of S. cerevisiae;lane 2, Eco RI digest of chromosomal DNA. B–D shows detected pno-containing band on the lane 2 by ECL Direct detection system.

Plasmids and localization of PNO gene quence similarity with GMC oxidoreductases. GMC M. luteolum YK-1 contained 4 probable plasmid oxidoreductases are FAD ‰avoproteins oxidoreduc- DNAs the approximate lengths of which were 150 kb, tases. These enzymes are proteins of size ranging 350 kb, 630 kb, and 910 kb (Fig. 5A). A PN 4- from 532 (4-nitrobenzyl alcohol dehydrogenase) to oxidase-encoding gene ( pno) was detected on the 664 (methanol oxidase) amino acid residues. Thus, DNA with a large molecular weight corresponding to PN 4-oxidase has the shortest amino acid sequence in chromosomal DNA. The Eco RI digest of the chro- the GMC oxidoreductase family so far reported. In mosomal DNA showed broad DNA bands (Fig. 5B). this context, examination of the amino acid sequence A single DNA band with an approximate length of of PN 4-oxidase in PROSITE showed that it has only 9 kb in the digest was stained with the probe, showing one of the signature patterns of GMC oxidoreduc- that the pno gene exists as one copy in the fragment tases at 254–268 (GSLESPALLMRSGIG): the se- of chromosomal DNA. quence corresponding to signature pattern 1 was not found. Thus, presence of both signature patterns in a Discussion given protein is not necessarily required for classiˆca- tion as a GMC oxidoreductase. PN 4-oxidase has been partially puriˆed from Pyridoxamine (pyridoxine) 5?-phosphate oxidase Pseudomonas MA-1, and its catalytic properties have (EC 1.4.3.5) catalyzes oxidation of pyridoxine 5?- been reported.3) The Pseudomonas enzyme was also phosphate to pyridoxal 5?-phosphate: the 4-hydroxy- FAD-dependent and showed the same optimum pH lmethyl group of pyridoxine moiety is oxidized as the as that of the Microbacterium enzyme. Both enzymes reaction by PN 4-oxidase. However, PN 4-oxidase showed a similar a‹nity for PN: the Pseudomonas and pyridoxamine 5?-phosphate oxidase, (which is a enzyme had a Km of 43 mM for PN. However, the dimeric enzyme with a molecular mass of Microbacterium enzyme showed about 17-fold 50,000–60,00021)) do not show similarity in their higher a‹nity for FAD than the former enzyme: the primary structures. Pseudomonas enzyme had a Km of 2.7 mM for FAD. M. luteolum YK-1 has the ˆrst enzyme of PN Thishigha‹nityforFADwassimilartothatofPN degradation pathway I, which was characterized by 5-oxidase from Arthrobacter Cr-7,2) which catalyzes Snell and a co-worker. In the pathway I, the second the oxidation of PN to isopyridoxal. All of the en- reaction is catalyzed by pyridoxal dehydrogenase (EC zymes show high and low substrate speciˆcities for 1.1.1.107). We have puriˆed the second enzyme from hydrogen donor and receptor substrates, respec- M. luteolum YK-1.4) The bacterium also showed tively. In contrast to the Microbacterium enzyme, pyridoxamine-pyruvate aminotransferase (EC which was a monomeric enzyme, PN 5- oxidase was 2.6.1.30) activity (data not shown). The results sug- dimeric: neither the molecular weight nor subunit gested that M. luteolum YK-1 degrades PN through structure of the Pseudomonas enzyme is known. the same or a closely similar pathway as the pathway PN 4-oxidase shared a number of regions of se- I. Elucidation of other genes encoding the enzymes in 1030 Y. KANEDA et al. the pathway of M. luteolum YK-1isunderway. aminophenazone chromogenic system in direct A PN 4-oxidase-encoding gene was found on a enzymic assay of uric acid in serum and urine. Clin. chromosomal DNA of M. luteolum YK-1. The en- Chem., 26, 227–231 (1980). zyme showed a high similarity to the mll6785 gene 8) Bradford, M. M., A rapid and sensitive method for product (a probable dehydrogenase) of a nitrogen- the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. ˆxing symbiotic bacterium, M. loti.Thecompletege- Chem., 72, 248–254 (1976). nome structure, including two megaplasmids, of M. 9) Laemmli, U. K., Cleavage of structural proteins 16) loti has been reported. The mll6785 gene was found during the assembly of the head of bacteriophage T4. at coordinates of 5585874–5584915 (a complement Nature, 227, 680–685 (1970). direction) of a chromosomal DNA of the bacterium. 10) Saito, H. and Miura, K., Preparation of transform- Predicted functions of other genes around the ing deoxyribonucleic acid by phenol treatment. mll6785 gene were examined. Interestingly, the mlr Biochim. Biophys. Acta, 72, 619–629 (1963). 6788 gene coordinated at 5587739–5588875 (a direct 11) Nakano, M., Morita, T., Yamamoto, T., Sano, H., direction) coded 2-methyl-3-hydroxypyridine-5- Ashiuchi, M., Masui, R., Kuramitsu, S., and Yagi, carboxylic acid oxygenase (EC 1.14.12.4) which T., Puriˆcation, molecular cloning, and catalytic catalyzes the 7th reaction step in PN degradation by activity of Schizosaccharomyces pombe pyridoxal reductase. A possible additional family in the aldo- pathway I. The oxygenase has been cloned from keto reductase superfamily. J. Biol. 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