This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Melnick, J.
Right arrow Articles by Begley, T. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Melnick, J.
Right arrow Articles by Begley, T. P.

 Previous Article  |  Next Article 

Journal of Bacteriology, June 2004, p. 3660-3662, Vol. 186, No. 11
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.11.3660-3662.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification of the Two Missing Bacterial Genes Involved in Thiamine Salvage: Thiamine Pyrophosphokinase and Thiamine Kinase

Jonathan Melnick,1 Ewa Lis,1 Joo-Heon Park,1 Cynthia Kinsland,1 Hirotada Mori,2 Tomoya Baba,3 John Perkins,4 Ghislain Schyns,4 Olga Vassieva,5 Andrei Osterman,5 and Tadhg P. Begley1*

Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853,1 Nara Institute of Science and Technology, Ikoma, Nara 630-0101,2 Institute of Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0035, Japan,3 Abteilung VFB, Roche Vitamins Ltd., CH-4070 Basel, Switzerland,4 Integrated Genomics, Chicago, Illinois 606125

Received 5 September 2003/ Accepted 17 February 2004


arrow
ABSTRACT
 
The genes encoding thiamine kinase in Escherichia coli (ycfN) and thiamine pyrophosphokinase in Bacillus subtilis (yloS) have been identified. This study completes the identification of the thiamine salvage enzymes in bacteria.


arrow
TEXT
 
Thiamine pyrophosphate biosynthesis occurs by a complex multistep pathway (2). Therefore, bacteria have evolved a set of salvage kinases to utilize dephosphorylated intermediates present in growth medium (Fig. 1). Pyrimidine alcohol and thiazole alcohol salvage requires pyrimidine alcohol kinase (ThiD) and thiazole kinase (ThiM), two previously characterized enzymes (3, 4, 7, 9). Thiamine salvage occurs in two steps, with thiamine kinase (5) catalyzing the formation of thiamine phosphate, and thiamine phosphate kinase (ThiL) (12), a biosynthetic enzyme, catalyzing the conversion of this intermediate to thiamine pyrophosphate. An alternative to this two-step thiamine salvage pathway has been found in Saccharomyces cerevisiae, where thiamine pyrophosphokinase (THI80) catalyzes the direct conversion of thiamine to thiamine pyrophosphate (1, 6). This single-step thiamine salvage pathway was previously thought to occur only in eukaryotes. Here we describe the characterization of the genes encoding thiamine kinase from Escherichia coli (ycfN) and thiamine pyrophosphokinase from Bacillus subtilis (yloS). This study reveals different thiamine salvage strategies in the two bacteria and completes the identification of the thiamine salvage enzymes in bacteria.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 1. Thiamine salvage pathway in E. coli and B. subtilis. The thiamine kinase and thiamine pyrophosphokinase described in this paper are shown in bold. Thiamine kinase is found in E. coli but not in B. subtilis, and thiamine pyrophosphokinase is found in B. subtilis but not in E. coli. HMP, pyrimidine alcohol; Thi-PP, thiamine pyrophosphate; OP, phosphate; OPP, pyrophosphate.

The thiamine kinase gene in E. coli was previously mapped between fabD and purB and was subcloned on a 2.6-kb fragment containing ClaI, MluI, SalI, HpaI, and NruI restriction sites (T. Fujio, M. Hayashi, A. Iida, T. Nishi, and T. Hagihara, 1991, Thiamine phosphates and their enzymic manufacture using recombinant Escherichia, European Patent Application EP 417953 A1). Of these, the MluI, SalI, and HpaI sites were contained within the gene. This information was sufficient to identify a region on the E. coli chromosome that contained a ClaI site, two closely spaced MluI sites, a SalI site, two closely spaced HpaI sites, and an NruI site on a 2.9-kb region (bases 1161161 to 1164160). The MluI, SalI, and HpaI sites were all within the ycfN gene, strongly suggesting that ycfN is the thiamine kinase gene. A protein BLAST search of the YcfN sequence was uninformative, indicating only weak sequence similarity to a glycosidase.

The E. coli ycfN gene was readily overexpressed in E. coli as a His10-tagged 34-kDa protein. This protein was present primarily in inclusion bodies, was unstable, and lost all activity during purification (Fig. 2, lanes 1 to 3). Assays to test the functional prediction were therefore carried out using cell extract and measuring thiamine phosphate formation after its conversion to the highly fluorescent thiochrome phosphate. High-performance liquid chromatography (HPLC) analysis of this reaction mixture (8) clearly demonstrated that YcfN catalyzed the phosphorylation of thiamine (37% conversion of a 3.3 µM solution in 1 h), while cell extract from the host strain did not show this activity.



View larger version (70K):
[in this window]
[in a new window]
  		FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of YcfN and YloS overexpression. Lane 1, total protein from the cell extract of the YcfN overexpression strain before induction; lane 2, total protein from the cell extract of the YcfN overexpression strain after induction; lane 3, soluble protein from the cell extract of the YcfN overexpression strain after induction; lane 4, total protein from the cell extract of the YloS overexpression strain before induction; lane 5, total protein from the cell extract of the YloS overexpression strain after induction; lane 6, soluble protein from the cell extract of the YloS overexpression strain after induction; lane 7, YloS after purification on a HisBind column.

B. subtilis can also salvage thiamine but does not contain a YcfN ortholog. The absence of this ortholog suggests that this microorganism may utilize a different strategy for thiamine salvage such as that found, for example, in S. cerevisiae, where thiamine is directly pyrophosphorylated (6). A protein BLAST search, using the S. cerevisiae thiamine pyrophosphokinase amino acid sequence (THI80), revealed that B. subtilis contains a protein (YloS) of previously unidentified function with low similarity to this sequence (17% sequence identity over 262 residues). To test the function of YloS, the gene was overexpressed in E. coli as a His6-tagged 24.5-kDa soluble protein and readily purified on a HisBind resin (Fig. 2, lanes 4 to 7). YloS was assayed for thiamine pyrophosphokinase activity by treating thiamine and ATP with the enzyme and assaying for thiamine pyrophosphate formation after its conversion to the highly fluorescent thiochrome pyrophosphate. HPLC analysis of the reaction mixture clearly demonstrated the presence of thiamine pyrophosphate (30% conversion of a 300 µM solution in 1 h). A continuous assay (9), based on the coupling of AMP production to NADH consumption, was used to determine the kinetic parameters of the enzyme (kcat, 0.2 s–1; Km of thiamine, 20 µM; Km of ATP, 1 mM).

The functional assignment of yloS and ycfN completes the identification of the thiamine salvage genes in bacteria. These genes show a surprisingly narrow distribution. YloS orthologs are found only in bacilli and lactobacilli, and YcfN orthologs are found only in the {gamma}-proteobacteria. Neither gene clusters with other thiamine biosynthetic genes. Based on these functional assignments, we propose that YcfN be renamed ThiK and that YloS be renamed ThiN.

Standard methods were used for DNA restriction endonuclease digestion, ligation, and transformation (11). PCR-derived DNA was sequenced and shown to contain no errors. The yloS gene was PCR amplified from B. subtilis 168 genomic DNA and overexpressed using pET28a in E. coli DH5{alpha} grown at 37°C in Luria-Bertani medium. The overexpressed protein was soluble and was purified on a HisBind column (Novagen). The ycfN gene was PCR amplified from E. coli genomic DNA and overexpressed using pET-16b(+) in E. coli DH5{alpha} grown at 37°C in Luria-Bertani medium.

YcfN and YloS were assayed for thiamine kinase and thiamine pyrophosphokinase activity, respectively, with the thiochrome assay (8, 10). For the thiamine pyrophosphokinase assay, YloS (51 µg, pure), thiamine alcohol (300 µM), ATP (300 µM), MgCl2 (2.2 mM), and KCl (11 mM) were incubated in 100 µl of 100 mM Tris HCl (pH 8) buffer. For the thiamine kinase assay, YcfN cell extract (10 µl of a 4.8-mg/ml solution) was added to a solution of thiamine (3 µM) and ATP (5 mM) in 290 µl of 0.1 M Tris (pH 8)-5 mM MgCl2. The reaction mixtures were incubated at 37°C, and 50-µl aliquots were removed after 0 and 60 min and quenched with 50 µl of 10% trichloroacetic acid. The denatured protein was removed by centrifugation (16,000 x g, 3 min), and 50 µl of 4 M potassium acetate followed by 50 µl K3Fe(CN)6 (30 mg/ml in 7 M NaOH) was added to the supernatant. The resulting solution was mixed by vortexing. After 1 min, 58 µl of 6 M HCl was added to quench the reaction. The resulting mixture was analyzed by HPLC (8).

The kinetic parameters for YloS were determined by using a coupled assay involving NADH consumption (9). A stock solution containing KCl (112 mg), MgCl2 (61 mg), NADH (4.3 mg), phosphoenolpyruvate (5.7 mg), pyruvate kinase (2.3 mg), lactate dehydrogenase (1.1 mg), and myokinase (1 mg) in 15 ml of 100 mM Tris HCl (pH 8) was prepared. Aliquots (250 µl) of this solution were placed in a quartz cuvette and diluted with 250 µl of 100 mM Tris HCl (pH 8) containing various concentrations of ATP and thiamine alcohol. The reaction was initiated by adding YloS (5 µl of a 10.1-mg/ml solution), and the consumption of NADH was measured by monitoring the decrease in absorbance at 340 nm ({varepsilon}340,NADH = 4.3 cm–1 mM–1; 2 NADH were consumed for each thiamine pyrophosphate produced).


arrow
ACKNOWLEDGMENTS
 
This research was supported by a grant from NIH to T.P.B. (DK44083) and by a gift from Hoffmann-La Roche.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853. Phone: (607) 255-7133. Fax: (607) 255-4137. E-mail: tpb2{at}cornell.edu. Back


arrow
REFERENCES
 
    1
  1. Baker, L. J., J. A. Dorocke, R. A. Harris, and D. E. Timm. 2001. The crystal structure of yeast thiamin pyrophosphokinase. Structure 9:539-546.[Medline]
    2. 2
  2. Begley, T. P., D. M. Downs, S. E. Ealick, F. W. McLafferty, A. P. Van Loon, S. Taylor, N. Campobasso, H.-J. Chiu, C. Kinsland, J. J. Reddick, and J. Xi. 1999. Thiamin biosynthesis in prokaryotes. Arch. Microbiol. 171:293-300.[CrossRef][Medline]
    3. 3
  3. Campobasso, N., I. I. Mathews, T. P. Begley, and S. E. Ealick. 2000. Crystal structure of 4-methyl-5-beta-hydroxyethylthiazole kinase from Bacillus subtilis at 1.5 Å resolution. Biochemistry 39:7868-7877.[CrossRef][Medline]
    4. 4
  4. Cheng, G., E. M. Bennett, T. P. Begley, and S. E. Ealick. 2002. Crystal structure of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate kinase from Salmonella typhimurium at 2.3 Å resolution. Structure 10:225-235.[Medline]
    5. 5
  5. Imamura, N., and H. Nakayama. 1982. thiK and thiL loci of Escherichia coli. J. Bacteriol. 151:708-717.[Abstract/Free Full Text]
    6. 6
  6. Nosaka, K., Y. Kaneko, H. Nishimura, and A. Iwashima. 1993. Isolation and characterization of a thiamin pyrophosphokinase gene, THI80, from Saccharomyces cerevisiae. J. Biol. Chem. 268:17440-17447.[Abstract/Free Full Text]
    7. 7
  7. Park, J.-H., K. Burns, C. Kinsland, and T. P. Begley. 2004. Characterization of two kinases involved in thiamine pyrophosphate and pyridoxal phosphate biosynthesis in Bacillus subtilis: 4-amino-5-hydroxymethyl-2-methylpyrimidine kinase and pyridoxal kinase. J. Bacteriol. 186:1571-1573.[Abstract/Free Full Text]
    8. 8
  8. Park, J.-H., P. C. Dorrestein, H. Zhai, C. Kinsland, F. W. McLafferty, and T. P. Begley. 2003. The biosynthesis of the thiazole moiety of thiamin pyrophosphate (vitamin B1). Biochemistry 42:12430-12438.[CrossRef][Medline]
    9. 9
  9. Reddick, J., C. Kinsland, R. Nicewonger, T. Christian, D. Downs, M. Winkler, and T. P. Begley. 1998. Overexpression, purification and characterization of two pyrimidine kinases involved in the biosynthesis of thiamin. Tetrahedron 54:15983-15991.[CrossRef]
    10. 10
  10. Reddick, J. J., R. Nicewonger, and T. P. Begley. 2001. Mechanistic studies on thiamin phosphate synthase: evidence for a dissociative mechanism. Biochemistry 40:10095-10102.[CrossRef][Medline]
    11. 11
  11. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
    12. 12
  12. Webb, E., and D. Downs. 1997. Characterization of thiL, encoding thiamin-monophosphate kinase, in Salmonella typhimurium. J. Biol. Chem. 272:15702-15707.[Abstract/Free Full Text]

Journal of Bacteriology, June 2004, p. 3660-3662, Vol. 186, No. 11
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.11.3660-3662.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Eudes, A., Erkens, G. B., Slotboom, D. J., Rodionov, D. A., Naponelli, V., Hanson, A. D. (2008). Identification of Genes Encoding the Folate- and Thiamine-Binding Membrane Proteins in Firmicutes. J. Bacteriol. 190: 7591-7594 [Abstract] [Full Text]  
  • Zayas, C. L., Claas, K., Escalante-Semerena, J. C. (2007). The CbiB Protein of Salmonella enterica Is an Integral Membrane Protein Involved in the Last Step of the De Novo Corrin Ring Biosynthetic Pathway. J. Bacteriol. 189: 7697-7708 [Abstract] [Full Text]  
  • Otte, M. M., Woodson, J. D., Escalante-Semerena, J. C. (2007). The Thiamine Kinase (YcfN) Enzyme Plays a Minor but Significant Role in Cobinamide Salvaging in Salmonella enterica. J. Bacteriol. 189: 7310-7315 [Abstract] [Full Text]  
  • Karunakaran, R., Ebert, K., Harvey, S., Leonard, M. E., Ramachandran, V., Poole, P. S. (2006). Thiamine Is Synthesized by a Salvage Pathway in Rhizobium leguminosarum bv. viciae Strain 3841.. J. Bacteriol. 188: 6661-6668 [Abstract] [Full Text]  
  • Schyns, G., Potot, S., Geng, Y., Barbosa, T. M., Henriques, A., Perkins, J. B. (2005). Isolation and Characterization of New Thiamine-Deregulated Mutants of Bacillus subtilis. J. Bacteriol. 187: 8127-8136 [Abstract] [Full Text]  
  • Kitagawa, M., Ara, T., Arifuzzaman, M., Ioka-Nakamichi, T., Inamoto, E., Toyonaga, H., Mori, H. (2005). 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 12: 291-299 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Melnick, J.
Right arrow Articles by Begley, T. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Melnick, J.
Right arrow Articles by Begley, T. P.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»