Biosci. Biotechnol. Biochem., 69 (12), 2277–2284, 2005 Purification and Characterization of Pyridoxine 50-Phosphate Phosphatase from Sinorhizobium meliloti y Masaaki TAZOE, ,* Keiko ICHIKAWA,** and Tatsuo HOSHINO* Department of Applied Microbiology, Nippon Roche Research Center, Kamakura, Kanagawa 247-8530, Japan Received April 15, 2005; Accepted July 29, 2005 Here we report the purification and biochemical 1-deoxy-D-xylulose 5-phosphate. The former is synthe- characterization of a pyridoxine 50-phosphate phospha- sized from D-erythrose 4-phosphate in three reaction tase involved in the biosynthesis of pyridoxine in steps catalyzed by Epd, PdxB, and SerC (PdxF) proteins Sinorhizobium meliloti. The phosphatase was localized in that order.4–7) The latter is formed from pyruvate and in the cytoplasm and purified to electrophoretic homo- D-glyceraldehyde 3-phosphate by 1-deoxy-D-xylulose 5- geneity by a combination of EDTA/lysozyme treatment phosphate synthase (Dxs).8) The two intermediates are and five chromatography steps. Gel-filtration chroma- then combined by PdxA and PdxJ proteins to generate 0 tography with Sephacryl S-200 and SDS/PAGE dem- the first vitamin B6 compound: pyridoxine 5 -phosphate onstrated that the protein was a monomer with a (PNP).9) PNP is finally oxidized to the active form, PLP, molecular size of approximately 29 kDa. The protein by a PNP/PMP oxidase (PdxH).10) PLP and PMP are required divalent metal ions for pyridoxine 50-phos- easily interconverted by ubiquitous transaminases. phate phosphatase activity, and specifically catalyzed Sinorhizobium (formerly known as Rhizobium) meli- the removal of Pi from pyridoxine and pyridoxal 50- loti IFO 14782 produces large quantities of PN.11) Since phosphates at physiological pH (about 7.5). It was two compounds, 1-deoxy-D-xylulose (1-DX) and 4- inactive on pyridoxamine 50-phosphate and other phys- hydroxy-L-theronine (4-HT), have been found to act as iologically important phosphorylated compounds. The precursors of PN in a study of the biosynthetic pathway 12) enzyme had the same Michaelis constant (Km) of 385 M of vitamin B6 we examined the biosynthesis of PN for pyridoxine and pyridoxal 50-phosphates, but its from 1-DX and 4-HT in vitro,13) and purified the specific constant [maximum velocity ðVmaxÞ=Km] was enzymes involved in its synthesis from the two nearly 2.5 times higher for the former than for the precursors (M. Tazoe, K. Ichikawa, and T. Hoshino, latter. unpublished results). BLAST searches of the five purified enzymes required for the formation of PN from Key words: phosphatase; pyridoxine 50-phosphate phos- the two compounds identified two putative xylulose phatase; vitamin B6; pyridoxine; Sinorhi- and homoserine kinases, 4-phosphohydroxy-L-threonine zobium (Rhizobium) meliloti dehydrogenase (PdxA), PNP synthase (PdxJ), and a conserved hypothetical protein, based on analysis of When pyridoxine (PN), which is also known as the chromosome sequence of S. meliloti strain 1021.14) vitamin B6***, and other compounds in this family are This suggests that PN is formed in the following taken up by living organisms, they are converted into sequence of reactions (Fig. 2): Initially, 1-DX and 4- 0 0 pyridoxal 5 -phosphate (PLP) and pyridoxamine 5 - HT are converted to 1-deoxy-D-xylulose 5-phosphate 15) phosphate (PMP). These act as cofactors of vitamin B6 and 4-phosphohydroxy-L-threonine by xylulose kinase enzymes that are active in the metabolism of amino and homoserine kinase16,17) respectively. The latter acids1,2) and deoxysugars.3) Many microorganisms (bac- product is then oxidized by PdxA, followed by con- teria, yeasts, fungi, and algae) and plants synthesize their densation with 1-deoxy-D-xylulose 5-phosphate by a own vitamin B6. The biosynthetic pathway in Esche- PdxJ protein to yield PNP, as it is in E. coli. The final richia coli is well understood (Fig. 1). It contains two step requires a hypothetical protein that dephospho- key intermediates, 4-phosphohydroxy-L-threonine and rylates PNP to PN and an enzyme with the required y To whom correspondence should be addressed. Tel/Fax: +81-42-739-8682; E-mail: [email protected] * Present address: Mycology and Metabolic Diversity Research Center, Tamagawa University Research Institute, Machida, Tokyo 194-8610, Japan ** Present address: Research Planning and Coordination Department, Kamakura Research Laboratories, Chugai Pharmaceutical Co., Ltd., 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan Abbreviations: 1-DX, 1-deoxy-D-xylulose; 4-HT, 4-hydroxy-L-threonine; p-NPP, p-nitrophenylphosphate; PLP, pyridoxal 50-phosphate; PMP, pyridoxamine 50-phosphate; PN, pyridoxine; PNP, pyridoxine 50-phosphate 0 *** In this paper, the term vitamin B6 is used to denote the six compounds, pyridoxine, pyridoxal, pyridoxamine, and their 5 -phosphate esters. 2278 M. TAZOE et al. Fig. 1. Currently Proposed Biosynthetic Pathway for Vitamin B6 in Escherichia coli. Materials and Methods Materials. ATP, ADP, AMP, malate, and phospho-L- serine were purchased from Wako Pure Chemical (Osaka, Japan). PN, PLP, PMP, p-nitrophenylphosphate (p-NPP), and most of the other biochemicals were obtained from Sigma Chemical (St. Louis, MO.). PNP was synthesized by reducing PLP with sodium borohy- dride according to the method of Stock et al.18) Most of the chromatographic media and prepacked columns were purchased from Amersham Biosciences (Uppsala, Sweden), and Ether Toyopearl was purchased from Toyo Soda Manufacturing (Tokyo). All other chemicals were of reagent grade and were purchased from commercial sources. Microorganisms, media, and cell growth. S. meliloti Fig. 2. Proposed Pathway for Pyridoxine from 1-Deoxy-D-xylulose IFO 14782 was cultured in a Pi-rich production medium and 4-Hydroxy-L-threonine in S. meliloti. composed of 4% glucose, 2% polypeptone, 0.2% yeast extract, 0.05% MgSO4.7H2O, 0.05% MnSO4.5H2O, properties, which is the subject of this paper. In and 0.001% FeSO4.7H2O (pH 6.8) at 28 C. The vitamin B6 biosynthesis in microorganisms, an enzyme Pi-limited chemically defined medium for the induction catalyzing dephosphorylation of PNP has not yet been of periplasmic marker proteins comprised the following: identified. With a view to understanding the dephos- 1% glucose and L-arginine, glycine, L-histidine, L- phorylation step in the biosynthesis of PN in S. meliloti isoleucine, L-leucine, L-lysine, L-methionine, L-threo- and our ultimate aim of gene engineering PN produc- nine, L-valine, L-aspartic acid, L-glutamic acid, L- tion, it is important to characterize the enzyme catalyz- phenylalanine, and L-tyrosine; pantothenic acid, inositol, ing the dephosphorylation of PNP. Here we show that nicotinic acid, thiamine, and biotin; and MnSO4.5H2O, this enzyme is a cytoplasmic PNP/PLP-specific phos- MgSO4.7H2O, CaCl2.2H2O, KCl, and KH2PO4, which phatase that is active as a monomer at physiological pH. was limited to 14 mg/l (pH 7.0). Sinorhizobium Pyridoxine 50-Phosphate Phosphatase 2279 Fractionation of cellular proteins. S. meliloti IFO dephosphorylase. The cell cake (59.5 g) formed by 14782 was grown in the production medium for 3 d. centrifugation of a 3-d culture broth (3.4-liter) in After harvesting by centrifugation, the cells were production medium was washed twice with 300 ml of subcultured in the chemically defined medium and in 0.85% NaCl and resuspended in 340 ml of buffer A. The the production medium for an additional 6 h. Then they cellular proteins were divided into periplasmic and were pelleted, washed twice with 0.85% NaCl, and cytoplasmic fractions using the procedure described suspended in buffer A [30 mM Tris–HCl (pH 8.0) con- above. taining 20% (w/v) sucrose and 1 mM EDTA]. Then Step 2. Q Sepharose column chromatography (I). The lysozyme (0.05%, w/v) was added at 23 C with slow lysate of the cytoplasmic fraction was centrifuged, and stirring for 20 min, and the suspensions were separated the supernatant was dialyzed against buffer B and into periplasmic proteins and cell pellets by centrifuga- applied to a Q Sepharose column (4:4 Â 17 cm). It was tion. The periplasmic fractions were dialyzed against developed with 600 ml of 400 mM KCl at a flow rate of buffer B [10 mM Tris–HCl (pH 7.5) containing 15% 0.57 ml/min. (w/v) sucrose, 1 mM dithiothreitol, and 0.1 mM phenyl- Step 3. Q Sepharose column rechromatography (II). methylsulfonyl fluoride], and alkaline phosphatase ac- The active fractions, which were dialyzed against tivity was measured by recording the hydrolysis of p- buffer B, were then applied to a Q Sepharose column NPP.19) The activity from the Pi-limited culture was (4:4 Â 12:5 cm). Elution was carried out at a flow rate of 1.56 mmol/min/mg protein, and that from the Pi-rich 0.72 ml/min with a linear gradient of KCl (0–400 mM)in culture was about 36-fold lower. When the cytoplasmic buffer B (total volume, 2,200 ml). marker enzyme malate dehydrogenase was assayed by Step 4. Ether Toyopearl column chromatography. The 20) monitoring the rate of oxidation of NADH (A340), no sample was dialyzed against buffer B and adjusted to activity was found in the periplasmic fractions of either 1.3 M (NH4)2SO4, and then applied to an Ether Toyo- culture. pearl column (2:5 Â 15 cm) previously equilibrated with To obtain the cytoplasmic proteins, pelleted cells buffer B containing 1.3 M (NH4)2SO4. Elution was were resuspended in buffer B and passed through a carried out at a flow rate of 0.7 ml/min with a linear French pressure cell, and the suspensions were centri- gradient of (NH4)2SO4 (1.3–0.5 M) in buffer B (total fuged at 37;500 Â g for 1 h. Cytoplasmic extracts of both volume, 1,000 ml).