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Purification and properties of 4-methyl-5-hydroxyethylthiazole kinase from Escherichia coli


Bioscience, Biotechnology, and Biochemistry

ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20

Purification and properties of 4-methyl-5hydroxyethylthiazole kinase from Escherichia coli
Yasushi Tani, Keisuke Kimura & Hisaaki Mihara
To cite this article: Yasushi Tani, Keisuke Kimura & Hisaaki Mihara (2015): Purification and properties of 4-methyl-5-hydroxyethylthiazole kinase from Escherichia coli, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2015.1104239 To link to this article: http://dx.doi.org/10.1080/09168451.2015.1104239

Published online: 03 Dec 2015.

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Bioscience, Biotechnology, and Biochemistry, 2015

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Puri?cation and properties of 4-methyl-5-hydroxyethylthiazole kinase from Escherichia coli
Yasushi Tani1,2, Keisuke Kimura1 and Hisaaki Mihara1,*
1

Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Japan; 2Ritsumeikan Global Innovation Research Organization, Ritsumeikan University, Kusatsu, Japan

Received August 11, 2015; accepted September 9, 2015 http://dx.doi.org/10.1080/09168451.2015.1104239

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4-Methyl-5-hydroxyethylthiazole kinase (ThiM) participates in thiamin biosynthesis as the key enzyme in its salvage pathway. We puri?ed and characterized ThiM from Escherichia coli. It has broad substrate speci?city toward various nucleotides and shows a preference for dATP as a phosphate donor over ATP. It is activated by divalent cations, and responds more strongly to Co2+ than to Mg2+. Key words: 4-methyl-5-hydroxyethylthiazole; ThiM; Escherichia coli; thiamin

Thiamin diphosphate is a cofactor for several key enzymes in carbohydrate metabolism, such as the pyruvate dehydrogenase complex, the 2-oxoglutarate dehydrogenase complex, and transketolase.1) Thiamin diphosphate is composed of thiazole and pyrimidine moieties, which are synthesized separately and then coupled to form thiamin phosphate and ?nally thiamin diphosphate (Fig. 1). The thiazole moiety is synthesized de novo through complex reactions from 1-deoxy-D-xylulose-5-phosphate, glycine, and cysteine, while the pyrimidine moiety is synthesized from 5-aminoimidazole ribonucleotide. In addition to de novo synthesis, the thiazole moiety is also synthesized by a salvage pathway catalyzed by 4-methyl-5-hydroxyethylthiazole (Thz) kinase (ThiM, EC 2.7.1.50), which is encoded by thiM in bacteria1) and plants2) and by the 3′ region of THI6 in Saccharomyces cerevisiae3) and Candida glabrata4) (Fig. 1). THI6 is a bifunctional enzyme comprising N-terminal and C-terminal domains corresponding to bacterial (and plant) thiamin phosphate synthase and ThiM, respectively. X-ray structures of ThiM from Bacillus subtilis,5) Enterococcus faecalis (PDB: 3dzv), Pyrococcus horikosii,6), and Staphylococcus aureus,7) as well as the THI6 enzyme of C. glabrata4) have been clari?ed, and some properties of ThiM from S. aureus8) and the THI6 from S. cerevisiae9) have been reported. In Escherichia coli, the thiM gene is located on the chromosome close to the thiD gene,10) which codes for
*Corresponding author. Email: mihara@fc.ritsumei.ac.jp
? 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry

a bifunctional enzyme containing both hydroxymethylpyrimidine kinase and phosphomethylpyrimidine kinase activities.11) The 5′-untranslated region of thiM mRNA contains a riboswitch that binds to thiamin diphosphate to regulate thiM translation.12,13) We obtained an ASKA clone JW2091 harboring pCA24N that encodes E. coli thiM gene with an N-terminal hexahistidine tag from the National BioResource Project (Japan).14) The cells were grown in LB medium containing 30 μg/mL chloramphenicol at 37 °C, and isopropyl β-D-1-thiogalactopyranoside was added at 0.1 mM, followed by further culture for 3 h. The cells were collected and suspended in 50 mM Tris HCl (pH 8.0) containing 300 mM NaCl, followed by sonication in the presence of 1% (v/v) Protease Inhibitor Cocktail for General Use (Nacalai Tesque, Kyoto, Japan) and 20 mM 2-mercaptoethanol. The lysate was loaded onto a Ni-NTA column (Qiagen, Vienna, Austria) equilibrated with 50 mM Tris HCl (pH 8.0) containing 300 mM NaCl, and the enzyme was eluted with 150 mM imidazole. Imidazole and NaCl were removed by repeated dilution with 10 mM Tris HCl (pH 8.0) and concentration with an Amicon Ultra-15 10K Centrifugal Filter Device (Millipore, USA). The ?nal enzyme preparation was supplemented with 1 mM dithiothreitol and stored at ?20 °C. Using the above procedure, we obtained a homogeneous preparation of E. coli ThiM. It showed the same molecular mass (28.1 kDa) on SDSPAGE as expected from the gene sequence. The activity of E. coli ThiM was determined at 37 °C using the standard assay mixture containing 100 mM Tris HCl (pH 7.5), 5 mM MgCl2, 5 mM ATP, and 0.5 mM Thz. After incubation for various time periods, the reaction mixture was mixed with half volume of 2 M HCl. The supernatant was neutralized with NaOH, and the 4-methyl-5-hydroxyethylthiazole phosphate (Thz-P) formed was detected by HPLC with a COSMOSIL 5C18-MS-II column (4.6 × 250 mm; Nacalai Tesque) under the following conditions: ?ow rate, 1 ml/min; detection, absorption at 261 nm; column temperature, 30 °C; elution, linear gradient with solutions A (0.1 M potassium phosphate buffer [pH 5.8]) and B (50% methanol in water) from 0% B (at 10 min)

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Y. Tani et al. Table 1. Substrate speci?city toward various nucleotides as a phosphate donor. Divalent metal ions added Phosphate donor ATP GTP CTP UTP TTP dATP dGTP dCTP dUTP
a

Mg2+

Co2+

Speci?c activitya (μmol/mg/min) 1.4 11 3.2 24 1.1 5.2 0.9 4.3 8.9 45 9.2 37 14 11 3.9 15 4.0 14

Assays were carried out at pH 10.0 and 50 °C.

Fig. 1.

The biosynthesis of thiamine diphosphate.

through 10% B (at 11 min) to 60% B (at 21 min), followed by isocratic elution with 60% B until 26 min. Thz-P was eluted at 18.1 min, whereas the substrate Thz was eluted at 25.4 min. The effect of pH on the ThiM reaction was examined using various buffers at 100 mM: sodium acetate (pH 4.0–6.0), potassium phosphate (pH 6.0–7.0), Tris HCl (pH 7.0–9.0), glycine NaOH (pH 9.0–9.5), and sodium carbonate (pH 9.5–11.5). The enzyme showed the highest activity at pH 11 and 70 °C. Therefore, we determined the speci?c activity of the ?nal preparation of E. coli ThiM at pH 10.5 and 70 °C to be about 34 μmol/mg/min. The values for other ThiMs have been reported: B. subtilis ThiM, 2.6 μmol/mg/min (pH 8.0)5); S. aureus ThiM, 4.8 μmol/mg/min (pH 7.5 and 37 °C)8); and ThiM activity of S. cerevisiae THI6, 0.5 μmol/mg/min (pH 7.5 and 37 °C).9) We examined the thermostability of E. coli ThiM by incubating it at 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90 °C for various durations (0–20 min) at pH 8.0 and by measuring the residual activity. No decrease in the activity was detected at temperatures lower than 50 °C, although about 60% of its original activity was lost at 60 °C after 20 min. Thus, E. coli ThiM is much more stable than S. cerevisiae THI6, because the ThiM activity of the yeast enzyme was decreased to about 20% of its original activity by incubation at 50 °C for 30 min.9) On the other hand, little is known about the thermostability of other ThiMs. We found that E. coli ThiM has broad speci?city toward various nucleotides that act as phosphate donors. TTP and GTP were better substrates than ATP, whereas CTP and UTP were less reactive than ATP under high-performance (sodium carbonate [pH 10.0] and 50 °C) conditions (Table 1). Notably, dATP was a much better substrate than ATP: enzyme activity in the

presence of dATP was more than six times better than that with ATP under the same conditions. S. cerevisiae THI6 resembles E. coli ThiM because various nucleotides such as dATP serve as substrates. However, the yeast enzyme differs from E. coli ThiM in that ATP is the best substrate for the yeast enzyme.9) Of the divalent cations, Co2+ was much more effective than Mg2+: more than seven times higher activity was observed when Mg2+ was replaced by Co2+ in under high-performance assay conditions. Thus, the best nucleotide and cation combination was TTP and Co2+, and the enzyme activity in the presence of this combination was more than 32 times higher than that achieved with the standard combination of ATP and Mg2+ (Table 1). The ThiM activity of S. cerevisiae THI6 is also known to be elevated in the presence of Co2+,9) but little information is available for bacterial ThiMs other than that reported here. X-ray structure of B. subtilis ThiM (formerly named ThiK) revealed that the substrate Thz interacts with Asn25, Val27, Ala33, Leu37, Pro43, Met45, Gly67, Thr68, and Thr194, which are located at the interface of subunits, while the phosphate of ATP interacts with Arg121, Thr168, Gly197, and Cys198.5) All these residues are highly conserved in E. coli ThiM: they correspond to Asn30, Val32, Ala38, Leu42, Pro48, Met50, Gly72, Thr73, and Val197; and Arg125, Thr171, Gly200, and Cys201, respectively, of the E. coli enzyme (Fig. 2). Of those residues, only Thr194 is replaced by Val in E. coli ThiM. Moreover, in B. subtilis ThiM, Mg2+ ion is coordinated by Asp94, Glu126, and Cys198. The corresponding residues in E. coli ThiM are Asp99, Glu130, and Cys201, showing complete conservation of the amino acids. Campobasso et al.5) proposed that Cys198 acts as the base to deprotonate the β hydroxyl group of Thz in B. subtilis ThiM. This residue is conserved as Cys201 in the E. coli enzyme. Campobasso et al.5) also showed that Cys198 is oxidized to form sul?nic acid if 2-mercaptoethanol is omitted from the enzyme solution during puri?cation. Thus, it may be possible that E. coli ThiM is readily inactivated by sulfhydryl reagents. Indeed, we found that E. coli ThiM lost about 70% of its original activity following addition of either iodoacetamide or iodoacetate (5 mM). This is probably due to the modi?cation of Cys201 by these reagents. Cys465 of the THI6 from

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ThiM from Escherichia coli

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Fig. 2. Sequence alignment of ThiMs from E. coli and B. subtilis. Notes: Sequence alignment is constructed by the Clustal W program. The asterisks indicate amino acid residues that are completely conserved in both enzymes. The positions of amino acid residues that are discussed in the text are indicated with the arrows.

S. cerevisiae occurring in its ThiM domain corresponds to the same cysteine residue. Kawasaki9) also reported that the yeast enzyme is readily inactivated by various sulfhydryl reagents such as p-chloromercuribenzoate. We determined the molecular mass of E. coli ThiM by gel ?ltration with COSMOSIL Diol-120-II (7.5 × 300 mm) and COSMOSIL Diol-300-II (7.5 × 300 mm) columns (Nacalai Tesque). The enzyme was eluted in a clear single peak with an estimated molecular mass of 56 kDa. The enzyme showed a single band with a molecular mass of 28 kDa on SDS PAGE. Therefore, the enzyme seems to have a dimeric structure. As described above, however, almost all important amino acid residues probably interacting with the substrate Thz are conserved in the E. coli enzyme as well, and they probably occur at the subunit interface. This strongly suggests that E. coli ThiM has the same subunit structure as other ThiMs, i.e. a homotrimer. Notably, S. aureus ThiM has been demonstrated to have a homotrimeric structure by X-ray crystallography,7) although it was originally thought to be a homodimer on the basis of the calculated molecular masses by gel ?ltration and SDS–PAGE.8) The discrepancy may be due to the unusual behavior of ThiMs in gel ?ltration chromatography. E. coli ThiM is characterized by its broad substrate speci?city toward various nucleotides. Recently, Tomita et al.15) demonstrated that pantoate kinase of Thermococcus kodakarensis shows broad nucleotide speci?city by using ATP, GTP, UTP, and CTP as substrates. However, it is not clear whether deoxynucleotides also serve as substrates of the pantoate kinase. The nucleotide binding site of B. subtilis ThiM has suf?cient space to accommodate nucleotides of different structures5) in the same manner as other ThiMs.4,6) E. coli ThiM probably has a large space to bind different nucleotides at the active site as well. However, one may wonder why dATP and dGTP are

much better as substrates than ATP. The X-ray structure of E. coli ThiM complexed with a deoxynucleotide would probably shed light on the mechanism of this unique substrate speci?city.

Authors’ contributions
Y.T., K.K., and H.M. designed the research plan; Y.T. and K.K. performed the experiments; Y.T., K.K., and H.M. analyzed the data; and Y.T. and H.M. wrote the manuscript.

Acknowledgment
The authors gratefully acknowledge Prof. Nobuyoshi Esaki, Kyoto University, for his excellent suggestions for improving this paper.

Disclosure statement
No potential con?ict of interest was reported by the authors.

Funding
This work was supported by the research grant (to H. M.) from the Ritsumeikan Global Innovation Research Organization (R-GIRO), Ritsumeikan University.

References
[1] Jurgenson CT, Begley TP, Ealick SE. The structural and biochemical foundations of thiamin biosynthesis. Annu. Rev. Biochem. 2009;78:569–603. [2] Yazdani M, Zallot R, Tunc-Ozdemir M, et al. Identi?cation of the thiamin salvage enzyme thiazole kinase in Arabidopsis and maize. Phytochemistry. 2013;94:68–73.

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Y. Tani et al. bifunctional enzyme in the thiamine biosynthetic pathway. J. Bacteriol. 1993;175:5153–5158. Mizote T, Nakayama H. The thiM locus and its relation to phosphorylation of hydroxylethylthiazole in Escherichia coli. J. Bacteriol. 1989;171:3228–3232. Mizote T, Tsuda M, Smith DD, et al. Cloning and characterization of the thiD/J gene of Escherichia coli encoding a thiaminsynthesizing bifunctional enzyme, hydroxymethylpyrimidine kinase/phosphomethylpyrimidine kinase. Microbiology. 1999;145:495–501. Serganov A, Polonskaia A, Phan AT, et al. Structural basis for gene regulation by a thiamine pyrophosphate-sensing riboswitch. Nature. 2006;441:1167–1171. Winkler W, Nahvi A, Breaker RR. Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature. 2002;419:952–956. Kitagawa M, Ara T, Arifuzzaman M, et al. Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res. 2006;12:291–299. Tomita H, Yokooji Y, Ishibashi T, et al. Biochemical characterization of pantoate kinase, a novel enzyme necessary for coenzyme a biosynthesis in the Archaea. J. Bacteriol. 2012;194:5434–5443.

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[3] Nosaka K, Nishimura H, Kawasaki Y, et al. Isolation and characterization of the THI6 gene encoding a bifunctional thiaminphosphate pyrophosphorylase/hydroxyethyl thiazole kinase from Saccharomyces cerevisiae. J. Biol. Chem. 1994;269:30510– 30516. [4] Paul D, Chatterjee A, Begley TP, et al. Domain organization in Candida glabrata THI6, a bifunctional enzyme required for thiamin biosynthesis in eukaryotes. Biochemistry. 2010;49:9922–9934. [5] Campobasso N, Mathews II, Begley TP, et al. Crystal structure of 4-methyl-5-β-hydroxyethylthiazole kinase from Bacillus subtilis at 1.5 ? resolution. Biochemistry. 2000;39:7868–7877. [6] Jeyakanthan J, Thamotharan S, Velmurugan D, et al. New structural insights and molecular-modelling studies of 4-methyl5-β-hydroxyethylthiazole kinase from Pyrococcus horikoshii OT3 (PhThiK). Acta Crystallogr. 2009;F65:978–986. [7] Drebes J, Perbandt M, Wrenger C, et al. Puri?cation, crystallization and preliminary X-ray diffraction analysis of ThiM from Staphylococcus aureus. Acta Crystallogr. 2011;F67:479–481. [8] Müller IB, Bergmann B, Groves MR, et al. The vitamin B1 metabolism of Staphylococcus aureus is controlled at enzymatic and transcriptional levels. PLoS ONE. 2009;4:e7656. [9] Kawasaki Y. Copuri?cation of hydroxyethylthiazole kinase and thiamine-phosphate pyrophosphorylase of Saccharomyces cerevisiae: characterization of hydroxyethylthiazole kinase as a

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