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Journal of Bacteriology, April 2008, p. 2629-2632, Vol. 190, No. 7
0021-9193/08/$08.00+0     doi:10.1128/JB.01722-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Novel Monofunctional Histidinol-Phosphate Phosphatase of the DDDD Superfamily of Phosphohydrolases{triangledown}

Hyun Sook Lee,{dagger} Yona Cho,{dagger} Jung-Hyun Lee, and Sung Gyun Kang*

Korea Ocean Research & Development Institute, Ansan P.O. Box 29, Seoul 425-600, Republic of Korea

Received 28 October 2007/ Accepted 11 January 2008


   ABSTRACT
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The TON_0887 gene was identified as the missing histidinol-phosphate phosphatase (HolPase) in the hyperthermophilic archaeon "Thermococcus onnurineus" NA1. The protein contained conserved motifs of the DDDD superfamily of phosphohydrolase, and the recombinantly expressed protein exhibited strong HolPase activity. In this study, we functionally assessed for the first time the monofunctional DDDD-type HolPase, which is organized in the gene cluster.


   TEXT
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Histidinol-phosphate phosphatase (HolPase; EC 3.1.3.15) catalyzes the eighth step in the histidine biosynthesis pathway, the dephosphorylation of histidinol-phosphate to histidinol, the direct precursor of histidine. HolPases belonging to the PHP (polymerase and histidinol phosphatase) superfamily in Bacillus subtilis, Saccharomyces cerevisiae, and Thermus thermophilus have been functionally characterized (15, 17, 19). The HolPases as members of the DDDD phosphohydrolase/phosphotransferase superfamily have been revealed to be bifunctional, with the C-terminal domain exhibiting imidazole glycerol-phosphate dehydratase (IGPD; EC 4.2.1.19) activity (8, 12, 16). In many organisms across all three kingdoms (bacteria, archaea, and eukaryotes), there is no report on the functional assessment of the monofunctional DDDD-type HolPase, whereas monofunctional IGPDs have been identified from fungi, plants, archaea, and some bacteria (11, 18, 20, 25).

In the sequenced archaeal genome of "Thermococcus onnurineus" NA1 (2; H. S. Lee and S. G. Kang, submitted for publication), most genes encoding the enzymes of the histidine biosynthesis pathway could be identified by sequence similarity with their counterparts, but the only gene encoding HolPase appeared to be missing. The analysis of the organization of his genes in "T. onnurineus" NA1 revealed that they are arranged in a compact cluster whose relative gene order, hisGDBHAF(IE)C, resembles that of the complete enterobacterial his operon, hisGDC(NB)HAF(IE) (1, 7) (Fig. 1). The same gene organization as that of "T. onnurineus" NA1 was also found in two bacterial genomes, those of Oceanobacillus iheyensis (26) and Lactobacillus plantarum (14), and in two hyperthermophilic-archaeal genomes, those of Thermococcus kodakarensis (10) and Pyrococcus furiosus (23), by the Sequence Similarity Database (SSDB) gene cluster search program (http://www.genome.jp) of the Kyoto Encyclopedia of Genes and Genomes (KEGG) (4, 13). The gene organization exhibited a translocation of the hisC gene with respect to the Escherichia coli arrangement, moving from downstream of hisD to downstream of his(IE) at the end of the cluster. Compact his gene clusters, with a hisC gene next to his(IE) or isolated from the cluster, have been detected in only a few genomes. In those cases, most monofunctional HolPases are not clustered with other his genes but are present elsewhere in the chromosome (6). It is peculiar, therefore, that the genes encoding PHP-type HolPases are located immediately downstream of hisG, encoding ATP phosphoribosyltransferase, which is next to hisZ, encoding the ATP phosphoribosyltransferase regulatory subunit in the cases of O. iheyensis and L. plantarum.


Figure 1
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  		FIG. 1. Alignment of gene clusters involved in the histidine biosynthesis pathway. Gene names abbreviated as one italic letter are those of E. coli, except N, which is named for a gene encoding monofunctional HolPase. The functional annotations of ORFs were based on those in the GenBank database. Contigs of "T. onnurineus" NA1, T. kodakarensis, P. furiosus, O. iheyensis, and L. plantarum were aligned according to their hisG genes. Shaded boxes represent the ORFs TON_0887, TK0251, and PF1666, conserved in "T. onnurineus" NA1, T. kodakarensis, and P. furiosus, respectively, and predicted to encode HolPases.

 
Because all three monofunctional IGPDs were present in the gene clusters of "T. onnurineus" NA1, T. kodakarensis, and P. furiosus, we attempted to search for the monofunctional HolPases. By the alignment of his genes as shown in Fig. 1, three open reading frames (ORFs) which are conserved in three genomes and located downstream of the hisC genes were found. They showed significant homology with each other (52.5 to 53.8% identity) (Fig. 2). The ORF of P. furiosus (PF1666) was not assigned a function (hypothetical protein), but those of "T. onnurineus" NA1 (TON_0887) and T. kodakarensis (TK0251) were predicted to be hydrolases belonging to the haloacid dehalogenase-like hydrolase (HAD) superfamily. A homology search of the ORFs by the Basic Local Alignment Search Tool (BLAST) program against the nonredundant protein database from the NCBI did not yield any significant match. Those three ORFs were the strongest candidate genes for the missing HolPase genes in three genomes, not because of their distinctive localization but because they are HAD members that contain three conserved motifs, including four invariant aspartate residues found in the DDDD superfamily (Fig. 2).


Figure 2
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  		FIG. 2. Alignment of the amino acid sequence of TON_0887 from "T. onnurineus" NA1 with those of orthologous proteins from T. kodakarensis and P. furiosus. The three motifs conserved in proteins belonging to the HAD superfamily are denoted with boxes, and four invariant aspartate residues are shown in bold. Identical residues among the three proteins are marked with asterisks, and residues with conserved substitutions and semiconserved substitutions are marked with colons and periods, respectively. The GenBank accession numbers of sequences are as follows: for TON_0887, EU487258; for TK0251, YP_182664; and for PF1666, NP_579395.

 
To determine whether the candidate gene, TON_0887, in "T. onnurineus" NA1 is a bona fide HolPase, we expressed the gene in Escherichia coli. The full-length TON_0887 gene was amplified by PCR with the primers sense-NdeI (5'-CGACCCGGCATATGAAGTGGATCATCTTCGACGTTG-3' [the italicized bases indicate the NdeI site]) and antisense-SalI (5'-CTCCACATGTCGACCCCCAATAAGTTCTCCAATAATTC-3' [the italicized bases indicate the SalI site]) from genomic DNA isolated by standard procedures (24). The amplified DNA fragments were digested with NdeI and SalI and then ligated into the NdeI/SalI-digested pET-24a(+) vector (Novagen, Madison, WI). E. coli Rosetta(DE3)pLysS (Stratagene, La Jolla, CA) was transformed with the constructed plasmid to express a recombinant protein. The His6 tag fusion protein was purified to homogeneity using a combination of Talon metal affinity column chromatography (BD Biosciences Clontech, Palo Alto, CA) and Superdex 200 10/300 GL column chromatography (Amersham Biosciences, Piscataway, NJ). The buffer of the protein was then exchanged with 50 mM Tris-HCl buffer (pH 8.0), which includes 10% glycerol, using Centricon YM-10 (Millipore, Bedford, MA). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the 30-kDa protein, which is the expected size of the fusion product comprising the 28.1-kDa protein and the 1.7-kDa peptide corresponding to the VDKLAAALEH6 (His6 tag) at the C-terminal region of the protein, was the major component of the purified sample (Fig. 3). Gel filtration under nondenaturing conditions showed a protein peak corresponding to a molecular mass of 39 kDa, implying that the protein exists as a monomer in solution (data not shown).


Figure 3
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  		FIG. 3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% polyacrylamide) of the purified TON_0887-derived protein. Lanes: 1, molecular mass standards (PageRuler protein ladder; Fermentas, Burlington, ON, Canada); 2, whole-cell lysate prior to isopropyl-β-D-thiogalactopyranoside (IPTG) induction; 3, whole-cell lysate after IPTG induction; 4, purified protein following metal affinity chromatography; 5, purified protein following gel filtration chromatography. The bands corresponding to the TON_0887-derived proteins are indicated with an arrow. Protein concentration was determined by the Bradford assay (5), and each of lanes 4 and 5 was loaded with 3 µg of protein.

 
To prove the functionality of the TON_0887-derived protein as the HolPase, the ability to release inorganic phosphate from histidinol-phosphate was investigated (3, 21). The protein displayed strong HolPase activity in the presence of Mg2+, Mn2+, Ni2+, Co2+, or Cu2+, as expected from the requirement of metal as a cofactor for catalysis, because TON_0887, as a member of the DDDD superfamily, possesses conserved residues in motifs I and III, which interact with a metal ion and are essential for activity (Fig. 2). The HolPase activity was buffer dependent and evaluated to be maximal at pH 6.5 (data not shown). However, the protein did not show any phosphatase activity against a general phosphatase substrate, p-nitrophenyl phosphate. Furthermore, with 26 various natural phosphatase substrates (Sigma, St. Louis, MO), including 17 ribo- and deoxyribonucleoside 5'- or 3'-mono-, di-, or triphosphates, 5 carbohydrate phosphates, 2 amino acid phosphates, and 2 small phosphates (pyrophosphate and acetyl phosphate), no significant activity was detected, except for AMP, fructose 6-phosphate, and phosphoserine. Kinetic parameters for the enzyme with histidinol-phosphate, AMP, fructose 6-phosphate, and phosphoserine were determined and are presented in Table 1. Km values for AMP, fructose 6-phosphate, and phosphoserine are 1 order of magnitude higher than that for histidinol-phosphate, which shows that HolPase has higher apparent substrate affinity for histidinol-phosphate. Furthermore, the enzyme displayed a 2- or 3-order-of-magnitude increase in catalytic efficiency (kcat/Km) with histidinol-phosphate compared with the efficiencies of the above-mentioned substrates, implying that the enzyme is highly specific for histidinol-phosphate. The kinetic parameters were affected by metal ions (Table 1). The enzyme showed a higher Km in the presence of Mg2+ or Ni2+ than in the presence of Co2+ or Mn2+. In the presence of Mg2+, Ni2+, or Co2+, the enzyme had similar kcat/Km ratios (3 x 106 to 5 x 106 M–1 s–1), whereas the activity was two- to threefold higher in the presence of Mn2+. Regardless of the metal ions, the substrate preference of the enzyme remained unchanged (data not shown). The kcat/Km ratio of the enzyme toward histidinol-phosphate in the presence of Mn2+ was comparable to that of the N-terminal domain of E. coli HisB, a bifunctional DDDD-type enzyme, as a result of the equal contributions of the respective kcat and Km values (22). TON_0887 was verified to play the same role as hisN, for which such enzymatic activity has actually been demonstrated, thus completing the his gene cluster, hisGDBHAF(IE)CN. The highly homologous genes PF1666 and TK0251 from P. furiosus and T. kodakarensis, respectively, are highly likely to encode HolPases, as is consistent with the previous report which designated PF1666 as hisN next to hisC at the end of the his gene cluster in P. furiosus (9). Gene disruption studies and biochemical characterization of these orthologues would fortify their putative activities.


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  		TABLE 1. Kinetic parameters for the hydrolysis of various substrates by HolPase at 80°C and pH 6.5

 
This study is the first functional assessment of the monofunctional DDDD-type HolPase. Furthermore, HolPases in "T. onnurineus" NA1, T. kodakarensis, and P. furiosus are the first indication of monofunctional DDDD-type HolPases being organized in the his gene cluster. For the gene organization hisGDBHAF(IE)CN, it is not easy to explain how hisN got into the final position at present. It is possible that hisN was translocated from the middle to the end of gene cluster along with hisC before hisN and hisB were fused to a single bifunctional polypeptide, or perhaps hisN was recruited in the already constructed cluster hisGDBHAF(IE)C, where hisC had already been translocated. There are only a few cases for the occurrence of hisN(Z)GDBHAF(IE)C, where hisN is a PHP-type HolPase, but no hisNGDBHAF(IE) or hisGDBHAF(IE)N has been detected until now. The gene evolution of hisC as well as that of hisN might explain the gene organization.

The comparative genome analysis of his gene clusters in the 31 finished genomes revealed that TON_0887 is very distinct, as it is conserved only in some Thermococcales, although the conservation patterns of most other genes were very similar (H. S. Lee and S. G. Kang, unpublished data), suggesting that orthologous HolPases in other archaea might possess novel sequences.

In conclusion, we have predicted the missing HolPase gene in the histidine biosynthesis pathway in the hyperthermophilic archaeon "T. onnurineus" NA1 by analyzing its primary sequence and gene organization and verified the functionality of the gene by expression, purification, and biochemical analyses, which indicate that it is the first monofunctional DDDD-type HolPase in the gene cluster.

Nucleotide sequence accession number. The TON_0887 sequence has been deposited in GenBank under accession no. EU487258.


   ACKNOWLEDGMENTS
 
We thank V. Jo Davisson (Purdue University) for providing us with histidinol-phosphate.

This work was supported by the KORDI in-house program (PE97802) and the Marine and Extreme Genome Research Center Program of the Ministry of Maritime Affairs and Fisheries, Republic of Korea, and by a Korea Research Foundation grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2006-532-C00011).


   FOOTNOTES
 
* Corresponding author. Mailing address: Korea Ocean Research & Development Institute, Ansan P.O. Box 29, Seoul 425-600, Republic of Korea. Phone: 82-31-400-6241. Fax: 82-31-406-2495. E-mail: sgkang{at}kordi.re.kr Back

{triangledown} Published ahead of print on 25 January 2008. Back

{dagger} These authors contributed equally to this work. Back


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Journal of Bacteriology, April 2008, p. 2629-2632, Vol. 190, No. 7
0021-9193/08/$08.00+0     doi:10.1128/JB.01722-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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