Journal of Bacteriology, March 2004, p . 1571-1573, Vol . 186, No . 5

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

Joo-Heon Park, Kristin Burns, Cynthia Kinsland, and Tadhg P . Begley^*

Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853

Received 29 May 2003/ Accepted 10 October 2003

 
Two Bacillus subtilis genes encoding two proteins [currently annotated ThiD and YjbV] were overexpressed and characterized.YjbV has 4-amino-5-hydroxymethyl-2-methylpyrimidine and 4-amino-5-hydroxymethyl-2-methylpyrimidinepyrophosphate kinase activity and should be reannotated ThiD,and B . subtilis ThiD has pyridoxine, pyridoxal, and pyridoxaminekinase activity and should be reannotated PdxK.

 
The biosynthesis of thiamine pyrophosphate [TPP] involves the coupling of 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate [HMP-PP] and 4-methyl-5-ß-hydroxyethylthiazole phosphate[Thz-P] to form thiamine phosphate followed by a final phosphorylation[1] . In addition to the de novo biosynthesis, microorganismshave developed several salvage pathways for the biosynthesisof TPP [Table 1] . Thiamine from the growth medium is either phosphorylated by thiamine kinase or pyrophosphorylated by thiamine pyrophosphokinase [J . Melnick, E . Lis, J.-H . Park, H . Mori,C . Kinsland, J . Perkins, G . Schyns, A . Osterman, and T . P . Begley, submitted for publication] . The pyrimidine and thiazole components can also be salvaged: thiazole is phosphorylated by thiazolekinase [2, 4, 6], HMP is phosphorylated to HMP-P by both ThiDand PdxK [3, 7, 10], and the phosphorylation of HMP-P is catalyzed by ThiD [5, 6, 7] . Thus, ThiD has both a biosynthetic and asalvage function in thiamine biosynthesis . PdxK is able to phosphorylatea broad range of substrates, including HMP, pyridoxal [PL],pyridoxamine [PM], and pyridoxine [PN], and is a salvage enzymein the biosynthesis of thiamine as well as that of PL phosphate[PLP].

 
A search of the Bacillus subtilis genomic database [http://genolist.pasteur.fr/SubtiList/index.html] shows homologues of Escherichia coli ThiD and PdxK named YjbV [1246149-1246961] and ThiD [3899983-3900795] . They are both 271-amino-acid proteins . yjbV is located immediately downstream of the thiOSGF operon that is involved in Thz-P biosynthesis, while thiD is not clustered with any of the thiamine or PLP biosynthetic genes . E . coli PdxK shows 24 and 25% sequence identity with B . subtilis YjbV and ThiD, respectively, and E . coli ThiDshows 41 and 35% identity with B . subtilis YjbV and ThiD, respectively.The level of sequence homology between these two proteins istoo high to allow the preferred substrate to be predicted foreither protein . However, the occurrence of yjbV in the thiazolebiosynthetic operon suggests that these proteins are incorrectlyannotated and that YjbV might function as the B . subtilis HMP/HMP-Pkinase . Here we report the overexpression of YjbV and ThiD fromB . subtilis and the identification of the substrate preferencesof the two proteins.

The amino acid sequences of E . coli ThiD and PdxK were obtained from the National Center for Biotechnology Information [http://www.ncbi.nlm.nih.gov/] and used with the SubtiList World Wide Web server for a BLAST search . For cloning B . subtilis thiD and yjbV, standard DNA restriction endonuclease digestion, ligation, and transformation methods were used [9] . Genomic DNA and plasmid DNA were purifiedwith a Wizard Plus SV genomic DNA kit and a DNA Miniprep kit,respectively [Promega] . DNA fragments were separated by agarose gel electrophoresis, excised, and purified with a QIAquick gel extraction kit [Qiagen] . pET-16b plasmid was obtained from Novagen. E . coli strain DH5 was used as a recipient for transformationduring plasmid construction and for plasmid propagation andstorage . E . coli BL21[DE3] was purchased from Novagen and usedas a host strain for the overexpression of the proteins . A PerkinElmer GeneAmp PCR System 2400 apparatus and Platinum Pfx DNApolymerase [Gibco Life Technologies] were used for PCR . B . subtilisCU1065 genomic DNA was used as a template for PCR . Primer synthesisand DNA sequencing were performed by the Bioresource Centerat Cornell University . Primers introduced NdeI and XhoI restriction enzyme sites at the 5' and 3' ends, respectively.

For the overexpression and purification of ThiD and YjbV, their corresponding overexpression plasmids were transformed intocompetent E . coli BL21[DE3] cells and the transformed cellswere grown at 37°C in Luria-Bertani medium containing 50mg of ampicillin/liter . To induce the overexpression of proteins,isopropyl-ß-D-thiogalactopyranoside [IPTG] was addedto the culture [when the optical density at 595 nm reached 0.6]to achieve a final concentration of 1 mM . Culture growth wascontinued for 8 h at 28°C, after which the cells were harvested and stored at -80°C until further use . The proteins were purified according to a Qiagen protocol for the purificationof His-tagged proteins . The eluted proteins were rapidly desaltedusing a PD-10 column [Amersham Pharmacia] because of instabilityunder high-salt concentrations and stored in 5% glycerol at-80°C . ThiD was soluble and stable in 50 mM Tris buffer[pH 8], but YjbV solutions rapidly became turbid . The resultsof sodium dodecyl sulfate-polyacrylamide gel electrophoresis[SDS-PAGE] with the purified proteins are shown in Fig . 1 . Althoughthe migration characteristics of the purified proteins weredifferent, their molecular weights were confirmed by mass spectrometry[data not shown].

 
The reaction mixtures for B . subtilis ThiD and YjbV enzymatic assays contained 1 mM ATP, 1 mM HMP, 2 mM MgCl₂, and 40 µg of enzyme in 100 µl of 50 mM Tris-HCl [pH 8] . After incubationat 37°C for 10 min, the reaction was quenched by the additionof 100 µl of 10% trichloroacetic acid and centrifugedto remove proteins . A total of 20 µl of the reaction mixturewas analyzed by high-pressure liquid chromatography [HPLC] [SupelcosilLC-18-T] [15- by 4.6-mm column] . The elution conditions wereas follows: flow rate, 1 ml/min; elution time, 0 to 20 min;elution buffer, 100% of 0.1 M potassium phosphate [pH 6.6].To conduct a competition assay, ThiD was incubated with allfour substrates [0.3 mM concentrations each of HMP, PL, PM,and PN] for 30 min under the conditions described above [exceptthat 2 mM ATP was used and the reaction mixture was analyzed by HPLC].

For kinetic studies, ADP produced by the kinase activity ofThiD or YjbV was assayed using a pyruvate kinase-lactate dehydrogenase-coupled system [which uses ADP and NADH as substrates] . The consumption of NADH by this coupled system can be measured by monitoringthe decrease in absorbance at 340 nm [7] . Pyruvate kinase, lactate dehydrogenase, phosphoenolpyruvate, NADH, and PL were purchased from Sigma . HMP was synthesized as previously described [8]. The assay mixture for the kinetic analysis of ThiD in the presence of HMP or PL contained saturating concentrations of ATP [5 mM], 30 to 400 µM HMP [or 30 to 300 µM PL], 10 mM MgCl₂,50 mM KCl, 0.2 mM NADH, 1 mM phosphoenolpyruvate, 8 units ofpyruvate kinase/ml, and 10 units of lactate dehydrogenase/mlin 0.6 ml of 50 mM Tris-HCl [pH 8] . Addition of ThiD to achievea final concentration of 6.7 µM initiated the reactions,which were then monitored over 5 min for NADH consumption at340 nm.

HPLC analysis of the reaction mixture containing B . subtilis YjbV showed the appearance of two new peaks corresponding to HMP-P and HMP-PP [Fig . 2A] . The reaction mixture containing B . subtilis ThiD showed only one pyrimidine product peak, which corresponds to HMP-P [Fig . 2B] . In addition to the phosphorylationof HMP, B . subtilis ThiD was able to phosphorylate PL, PM, andPN, producing PLP, PMP, and PNP, respectively . Under similarconditions, YjbV did not catalyze the phosphorylation of thesecompounds [data not shown] . A competition assay using the substratesof ThiD revealed a preference for PL followed by HMP, PN, andPM [8:2.4:1.1:1 product ratios] . The kinetic parameters forB . subtilis ThiD are shown in Table 2 . The kinetic parametersof B . subtilis YjbV could not be determined, because the reactionmixture became turbid immediately after the reaction began.

 
Overall our results indicate that B . subtilis YjbV has HMP/HMP-P kinase activity and should be reannotated ThiD [i.e., the name should be changed from YjbV to ThiD] and that B . subtilis ThiD has PN/PL/PM/HMP kinase activity and should be reannotated PdxK [i.e., from ThiD to PdxK].

 
This research was supported by a grant from NIH [DDK44083].

 
* 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@cornell.edu .

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