Journal of Bacteriology, September 2004, p . 6070-6076, Vol . 186, No . 18

Among Multiple Phosphomannomutase Gene Orthologues, Only One Gene Encodes a Protein with Phosphoglucomutase and Phosphomannomutase Activities in Thermococcus kodakaraensis

Naeem Rashid, Tamotsu Kanai, Haruyuki Atomi, and Tadayuki Imanaka^*

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, Japan

Received 11 April 2004/ Accepted 8 June 2004

 
Four orthologous genes [TK1108, TK1404, TK1777, and TK2185]that can be annotated as phosphomannomutase [PMM] genes [COG1109]have been identified in the genome of the hyperthermophilicarchaeon Thermococcus kodakaraensis KOD1 . We previously foundthat TK1777 actually encodes a phosphopentomutase . In orderto determine which of the remaining three orthologues encodesa phosphoglucomutase [PGM], we examined the PGM activity inT . kodakaraensis cells and identified the gene responsible forthis activity . Heterologous gene expression and purificationand characterization of the recombinant protein indicated thatTK1108 encoded a protein with high levels of PGM activity [690U mg^–1], along with high levels of PMM activity [401 Umg^–1] . Similar analyses of the remaining two orthologuesrevealed that their protein products exhibited neither PGM norPMM activity . PGM activity and transcription of TK1108 in T.kodakaraensis were found to be higher in cells grown on starchthan in cells grown on pyruvate . Our results clearly indicatethat, among the four PMM gene orthologues in T . kodakaraensis,only one gene, TK1108, actually encodes a protein with PGM andPMM activities.

 
Genome sequencing has provided an enormous amount of informationon the primary structure and number of genes in a particularorganism [5, 11] . Based on the assumption that genes with high levels of similarity encode proteins that have the same function,the presence or absence of various orthologues is often usedin estimating whether a specific metabolic pathway is present.This approach, however, has its limitations . When an orthologueof an expected enzyme is not found, the gene must be identifiedthrough classical biochemical and cloning methods [13, 14].On the other hand, when multiple orthologues are present on the genome, each gene product must be carefully examined in order to distinguish the enzymatic activities or functions inthe cell . Analyses of the expression levels of the genes alsocontribute to elucidating their functions [17].

We recently determined the entire genome sequence of a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 [unpublished data]. This strain was originally isolated from a solfatara on Kodakara Island, Kagoshima, Japan [2, 9] . It displays heterotrophic growthon a variety of organic substrates, such as amino acids, pyruvate,and starch, and we have taken an interest in the metabolic pathwaysinvolved in the assimilation of these carbon compounds . Throughgenome annotation, it has been possible to identify orthologuesfor a majority of the enzymes involved in glycolysis and gluconeogenesis,including the archaeal ADP-dependent glucose kinase, the ADP-dependentphosphofructokinase, and the archaeal class IA type fructose1,6-bisphosphate aldolase [7] . An orthologue of the key enzymeof gluconeogenesis, fructose 1,6-bisphosphatase, was not present,and a novel, archaeal-type fructose 1,6-bisphosphatase fromT . kodakaraensis was subsequently identified and characterized[14].

Although phosphoglucomutase [PGM] [EC 5.4.2.2] is not a memberof the glycolytic enzyme group, we also noticed that the geneencoding this enzyme has not been identified in Archaea . PGMcatalyzes the interconversion of glucose 6-phosphate and glucose1-phosphate, and it plays a vital role in carbohydrate metabolismin a wide range of microorganisms, as well as in plant and animalcells [6, 8, 10, 15] . From a catabolic point of view, PGM providesthe glycolytic intermediate glucose 6-phosphate from glucose1-phosphate, which is often the product of intracellular polysaccharidedegradation by various glycan phosphorylases [20] . A well-knownexample is the glycolytic reentry of glucose that has been storedas energy in the form of glycogen or trehalose [15] . On theother hand, glucose 1-phosphate is the precursor of sugar nucleotidesthat are necessary in the synthesis of various glucose-containing polysaccharides . Therefore, PGM also has an important biosynthetic role, supplying the glucose 1-phosphate from glucose 6-phosphatethat is produced through glycolysis or gluconeogenesis [15].

PGM activity has been detected in crude extracts of several archaeal strains [21, 22] . In the genome databases, many openreading frames have been annotated as genes encoding putativephosphomannomutases [PMMs] and can be considered likely candidatesto encode archaeal PGMs . However, most genomes harbor more thanone open reading frame designated a PMM gene . For example, Pyrococcusfuriosus, Pyrococcus abyssi, and Pyrococcus horikoshii havethree orthologues, while two orthologues have been found inthe Methanococcus jannaschii and Aeropyrum pernix genomes . Likethe genomes of other hyperthermophilic archaea, the T . kodakaraensisgenome harbors more than one open reading frame that is annotatedas a PMM gene; actually, it harbors four such open reading frames[TK1108, TK1404, TK1777, and TK2185] . While all four of theseopen reading frames are members of cluster 1109 of orthologousgenes [COG1109], it has been found previously that TK1777 actuallyencodes a phosphopentomutase [13] . This was unexpected, as allpreviously identified genes encoding phosphopentomutases weremembers of COG1015 and had a distinct primary structure . Therefore,as mentioned above, biochemical analyses of the remaining threeorthologues is necessary to accurately determine their individualfunctions . In this study, we obtained biochemical evidence thatallowed us to identify the true archaeal PGM gene in T . kodakaraensis.

 
Bacterial strains, plasmids, and media. T . kodakaraensis KOD1 was isolated from a solfataric hot springat a wharf on Kodakara Island, Kagoshima, Japan [2, 9] . T . kodakaraensiscells were grown in either a nutrient-rich MA-YT medium [13]or minimal ASW-AA medium [16]. Escherichia coli strain DH5 wasused for subcloning of the gene fragments and DNA manipulation. E . coli strain BL21[DE3] [Novagen, Madison, Wis.] was used as a host, and pET-21a [Novagen] was used as a vector for gene expression.

Chemicals and enzymes. Glucose 1-phosphate, glucose 6-phosphate, glucose 1,6-bisphosphate,mannose 1-phosphate, mannose 6-phosphate, fructose 1-phosphate,fructose 6-phosphate, glucosamine 1-phosphate, glucosamine 6-phosphate,N-acetylglucosamine 1-phosphate, N-acetylglucosamine 6-phosphate,2-deoxyribose 1-phosphate, 2-deoxyribose 5-phosphate, 3-phosphoglycericacid, 2-phosphoglyceric acid, 2,3-diphosphoglyceric acid, phosphoglucoisomerase, phosphomannoisomerase, glucose 6-phosphate dehydrogenase, enolase, pyruvate kinase, and lactate dehydrogenase were purchased from Sigma [St . Louis, Mo.] . Alcohol dehydrogenase, NADP, and NADHwere purchased from Oriental Yeast [Tokyo, Japan] . Deoxyribose5-phosphate aldolase [13] and glucosamine 6-phosphate deaminase [unpublished data] were purified from T . kodakaraensis . Other chemicals and components of various media were purchased from Wako Pure Chemicals [Osaka, Japan] or Nacalai Tesque [Kyoto,Japan].

DNA manipulation and sequencing. Restriction enzymes and DNA polymerase were purchased from Toyobo[Osaka, Japan] and Takara Shuzo [Kyoto, Japan] . Genomic andplasmid DNAs were isolated by using QIAGEN genomic and plasmidDNA isolation kits, respectively [QIAGEN, Hilden, Germany].DNA ligation was performed by using a DNA ligation kit [Toyobo].A QIAEX gel extraction kit [QIAGEN] was used to recover DNAfragments from agarose gels . DNA sequencing was performed with an ABI PRISM BigDye terminator cycle sequencing Ready Reaction kit [Applied Biosystems, Foster City, Calif.] . Database homology searches were performed by using the Basic Local Alignment Search Tool [BLAST] program [1] . Sequence analyses were performed by using DNASIS software [Hitachi Software, Yokohama, Japan] . Multiple-alignmentand phylogenetic analyses were performed by using the ClustalWprogram [18] provided by the DNA Data Bank of Japan [DDBJ].The phylogenetic tree was constructed by the neighbor-joiningmethod after alignment.

Partial purification of PGM activity from strain KOD1. T . kodakaraensis cells were cultivated at 85°C for 16 hin MA-YT medium [13] containing 0.5% soluble starch [Nacalai Tesque] . The cells were harvested and disrupted by sonication in ice water . All purification steps were performed at room temperature unless indicated otherwise . The membrane and cytosolic fractions from the cell lysate were separated by ultracentrifugation at 110,000 x g for 70 min at 4°C . The cytosolic fraction,containing the PGM activity, was loaded onto Resource Q [AmershamBiosciences, Little Chalfont, Buckinghamshire, United Kingdom]which had been equilibrated with buffer A [50 mM sodium phosphatebuffer, pH 7.0] . The proteins were eluted with a linear gradientof 0 to 1 M sodium chloride in buffer A . Then the fractionswith PGM activity were dialyzed against buffer A and loaded onto Mono Q HR 5/5 [Amersham Biosciences] which had been equilibrated with buffer A . The proteins were eluted with a linear gradient of 0 to 1 M sodium chloride in buffer A, and the fractions carrying PGM activity were dialyzed against 2 M ammonium sulfate andloaded onto Resource ISO [Amersham Biosciences] which had beenequilibrated with 2 M ammonium sulfate [pH 7.0] . The proteinswere eluted with a linear gradient of 2 to 0 M ammonium sulfate[pH 7.0] . Then the fractions carrying PGM activity were dialyzedagainst buffer A and loaded onto a hydroxyapatite column [Bio-ScaleCHT-I; Bio-Rad, Hercules, Calif.] . Fractions exhibiting PGMactivity were concentrated by using Centricon YM-30 [MilliporeCorporation, Bedford, Mass.] and were further purified on agel filtration column [Superdex 200 HR 10/30; Amersham Biosciences]which had been equilibrated with buffer A containing 150 mMsodium chloride . The protein concentration was determined witha bicinchoninic acid protein assay kit [Pierce, Rockford, Ill.]used according to the manufacturer's instructions; bovine serumalbumin was used as the standard . N-terminal amino acid residueswere determined with a protein sequencer [model 491 cLC; AppliedBiosystems] . The partially purified protein was also analyzedby matrix-assisted laser desorption ionization—time offlight mass spectrometry at Hitachi Science Systems [Hitachinaka,Japan].

Expression of the TK1108 gene and purification of the recombinant protein. NdeI and BamHI sites were introduced into the N and C termini of the TK1108 gene, respectively, by PCR . The DNA fragment was inserted into pET-21a at the NdeI and BamH I sites, resultingin pET-1108 . E . coli strain BL21[DE3] carrying pET-1108 wasgrown at 37°C in Luria-Bertani medium containing ampicillin[50 µg/ml] until an optical density at 660 nm of 0.5 wasreached . Gene expression was induced by addition of 0.2 mM [finalconcentration] isopropyl-ß-D-thiogalactopyranoside[IPTG], and the preparation was incubated for another 4 h at37°C . The cells were harvested by centrifugation at 6,000 x g for 10 min at 4°C and washed with 50 mM potassium phosphatebuffer [pH 7.0] . Then the cells were resuspended in the samebuffer and disrupted by sonication on ice . The supernatant aftercentrifugation [15,000 x g for 30 min at 4°C], containing the recombinant TK1108, was incubated at 85°C for 20 min. Heat-labile proteins were removed by centrifugation [15,000 x g for 30 min at 4°C] . Recombinant TK1108 was purifiedto homogeneity with Resource Q, Resource ISO, and Superdex 200HR10/30columns by using the methods as described above for partialpurification of PGM activity from T . kodakaraensis . RecombinantTK1404 and TK2185 were also expressed and purified by usingthe same method . The apparent homogeneity of the proteins was examined by sodium dodecyl sulfate [SDS]-polyacrylamide gel electrophoresis [PAGE] . Molecular mass estimates were obtainedby gel filtration [Superdex 200 HR 10/30] by using HMW and LMWgel filtration calibration kits [Amersham Biosciences].

Enzyme activity assay. PGM activity was measured by a discontinuous assay coupled withglucose 6-phosphate dehydrogenase . Formation of glucose 6-phosphatefrom glucose 1-phosphate was measured by monitoring NADPH formationwith glucose 6-phosphate dehydrogenase . The initial reactionmixture [200 µl] consisted of 100 mM Tris-HCl [pH 7.0],10 mM MgCl₂, 50 µM glucose 1,6-bisphosphate, 5 mM glucose1-phosphate, and enzyme solution . After incubation at the desiredtemperature for 1 min [for activity measurements, 60 to 95°C]or 5 min [30 to 50°C], the reaction mixture was cooled onice for 5 min, and the amount of glucose 6-phosphate producedwas measured by addition of 800 µl of water containing 0.5 mM NADP and 2 U of glucose 6-phosphate dehydrogenase . After incubation at 25°C for 3 min, the amount of NADPH was measuredat 340 nm . The amount of glucose 6-phosphate produced by theenzyme at 25°C during a 3-min period was subtracted . Theproduct formation was proportional to the incubation time underthese conditions . One unit was defined as the amount of activitythat produced 1 µmol of glucose 6-phosphate from glucose1-phosphate per min . All other enzyme activities were measuredas described previously [13].

When the effect of pH on the enzyme activity was examined, the reaction was carried out by using 100-µl reaction mixturescontaining the following buffers at a concentration of 20 mM:MES [morpholineethanesulfonic acid] buffer [pH 4.5 to 6.5],HEPES buffer [pH 6.5 to 7.5], and bicine buffer [pH 7.5 to 8.5].All of the buffers were prepared so that the pH reflected accuratevalues at 90°C . After the first reaction, 100 µl of1 M Tris-HCl [pH 8.0] was added to the reaction mixture to bringthe pH of the mixture to 8.0 [the optimal pH for the couplingenzyme] . To examine the effects of the various metal ions onenzyme activity, the first reaction mixture was incubated withthe metal cations . After incubation the mixture was cooled inice water, and as glucose 6-phosphate dehydrogenase is an Mg²⁺-dependentenzyme, the coupling reaction was initiated by adding 10 mMMg²⁺, NADP⁺, and the exogenous enzymes.

RNA isolation and dot blot analysis. RNA was isolated from cells grown in MA-YT medium supplementedwith either starch [0.5%] or sodium pyruvate [0.5%] . Cells wereharvested in the early log phase [optical density at 660 nm,0.1] . RNA was isolated with an RNeasy Midi kit [QIAGEN] . Fordot blot analysis, 1 µg of total RNA was denatured byheat treatment at 65°C for 15 min and spotted onto a nylonmembrane [Hybond-N+; Amersham Biosciences] . Digoxigenin labelingof DNA fragments, hybridization, and washing of the membraneswere performed according to the instructions of the manufacturer[Roche Diagnostics, Basel, Switzerland] . A DNA fragment correspondingto the entire TK1108 coding region was used as a probe . A controlexperiment was performed by using the DNA ligase gene from strainKOD1 as a probe.

Nucleotide sequence accession numbers. The nucleotide sequences of the TK1777, TK2185, TK1108, andTK1404 genes determined in this study have been deposited inthe DDBJ, EMBL, and GenBank DNA databases under accession numbers AB126239, AB126240, AB126241, and AB126242, respectively.

 
Presence of four putative PMM genes in the genome of T . kodakaraensis. Three of the four orthologues, TK1108 [encoding 456 amino acid residues with a molecular weight of 49,850], TK1777 [encoding450 amino acid residues with a molecular weight of 49,529],and TK2185 [encoding 449 amino acid residues with a molecularweight of 48,659], encoded proteins having similar molecularweights, while TK1404 encoded a smaller protein [236 amino acidswith a molecular weight of 26,728] . The three larger proteinsdisplayed high levels of similarity with one another [TK2185and TK1108, 44% identical; TK1777 and TK2185, 43% identical;TK1108 and TK1777, 38% identical] and also displayed equivalentlevels of similarity with the biochemically characterized PGMfrom E . coli [TK1777, 24%; TK2185, 24%; TK1108, 25%] . Two motifsconserved in various phosphosugar mutases [3, 19], TXSHNP containingthe active site serine residue and DXDXDR involved in metalbinding, were also conserved among these three proteins [Fig.1] . Therefore, it is quite difficult, if not impossible, todistinguish the functions of these enzymes from the sequencedata . We also noticed that highly related orthologues of TK2185,TK1108, and TK1404 are present in the genomes of P . furiosus,P . horikoshii, and P . abyssi . The high levels of similarityof the corresponding genes in the Thermococcales strains allowedus to classify these genes on the basis of their primary structure.A phylogenetic tree of the orthologues is shown in Fig . 2 . TK1777,the orthologue unique to T . kodakaraensis, actually encodeda phosphopentomutase and not a phosphohexomutase [13].

 
PGM activity in T . kodakaraensis and partial purification of PGM. In order to identify the gene[s] that actually encodes a PGM,the enzyme activity in extracts of T . kodakaraensis KOD1 cells grown in the presence of 0.5% yeast extract and 0.5% tryptonealong with 0.5% starch was examined . PGM activity was detectedin the cell extracts, and the specific activity was 0.8 U mg^–1.After the enzyme activity was determined, we partially purifiedthe PGM from the cell extract . PGM was purified 14-fold by anion-exchange, hydrophobic, hydroxyapatite, and gel filtration column chromatography. A 50-kDa protein was found to correspond well with the results of activity measurements through each purification step . Duringthis process, we did not observe PGM activity in fractions otherthan those used in the purification procedure . We analyzed theN-terminal amino acid sequence of the protein and found thatit corresponded to the sequence encoded by TK1777 . This wasunexpected, as this protein is a phosphopentomutase with onlytrace levels of PGM activity [13] . We then subjected the bandto matrix-assisted laser desorption ionization—time offlight mass spectroscopy [12] and found that it was a mixtureof two proteins, the proteins encoded by TK1777 and TK1108.Therefore, we analyzed TK1108.

Heterologous expression of the TK1108 gene and purification of the recombinant protein. We expressed the TK1108 gene in E . coli and obtained the recombinantprotein in a soluble form . The recombinant protein was purifiedto apparent homogeneity by heat treatment at 85°C for 20min, followed by anion-exchange, hydrophobic, and gel filtrationcolumn chromatography [Fig . 3] . The molecular mass of the proteinestimated by SDS-PAGE agreed with the value calculated fromthe deduced amino acid sequence . Furthermore, the six N-terminalamino acid residues of the purified recombinant protein wereidentical to the deduced amino acid sequence encoded by thegene . The purified protein exhibited high levels of PGM activity [420 U mg^–1] in the presence of 10 mM MgCl₂ and 5 mM glucose1-phosphate at 90°C and was designated PGM_Tk . Gel filtrationchromatography indicated that the molecular mass of PGM_Tk wasapproximately 210 kDa . Taking into account the molecular massof the subunit [49.8 kDa], this result indicates that PGM_Tkexists in a tetrameric form.

 
Effects of metal cations, pH, and temperature on PGM activity. Purified PGM_Tk was dialyzed against 25 mM Tris-HCl buffer [pH 8.0] containing 5 mM EDTA and used for further analysis . Activity measurements were performed by using a linked assay coupled with glucose 6-phosphate dehydrogenase . PGM_Tk did not display detectable PGM activity in the absence of metal ions, indicating that the activity was metal ion dependent . Addition of MgCl₂ [0.5 to 10 mM] led to significant levels of PGM activity, which was saturated at 1 mM [data not shown] . Besides Mg²⁺, we also found that Ni²⁺, Mn²⁺ and Zn²⁺ at a concentration of 1 mM couldalso support PGM activity, although to a lesser extent [Fig.4A].

 
We examined the effects of pH and temperature on the PGM_Tk activityin the presence of 10 mM Mg²⁺ . At a fixed temperature of 90°C,PGM_Tk displayed maximal activity at pH 7 . The temperature profileof the enzyme indicated that the optimal temperature was 90°Cunder our assay conditions [Fig . 4B] . The thermostability ofthe recombinant protein was monitored in the presence of 10mM Mg²⁺, and the protein was found to be highly stable evenat 100°C . The enzyme displayed a half-life of 85 min inboiling water [Fig . 4C] . A kinetic analysis was also carriedout, and PGM_Tk catalyzed the reaction with Michaelis-Mentenkinetics; the K_m with glucose 1-phosphate was 3.0 mM, and thek_cat was 575 s^–1 subunit^–1 at 90°C [Table 1].

 
Activity with various substrates. The mutase activities of PGM_Tk with various phosphorylated compounds[5 mM] were examined [Fig. 5] . Among the substrates, PGM_Tk exhibited high levels of mutase activity with glucose 1-phosphate and mannose 1-phosphate . The enzyme also displayed relatively low activity with deoxyribose 1-phosphate and glucosamine 1-phosphate [Fig . 5] . We carried out a kinetic analysis of the PMM activityof PGM_Tk and found that the reaction followed Michaelis-Mentenkinetics with a K_m of 3.2 mM and a k_cat of 330 s^–1 subunit^–1 at 90°C [Table 1] . We also examined the activity with 2-deoxyribose1-phosphate and obtained a K_m of 3.5 mM and a k_cat of 190 s^–1 subunit^–1 [Table 1] . No mutase activity was detected with fructose 1-phosphate, N-acetylglucosamine 1-phosphate, and 3-phosphoglycerate.

 
PGM activity in T . kodakaraensis KOD1. We examined the PGM activity in T . kodakaraensis KOD1 cellsgrown in a synthetic medium based on amino acids [ASW-AA medium][16] . This medium meets the minimal requirements for growthof strain KOD1 . To the ASW-AA medium, we also added either 0.5%starch [a glycolytic substrate] or 0.5% sodium pyruvate [a gluconeogenicsubstrate] . PGM activity was detected in all cell extracts,and the levels of activity were 0.16 ± 0.01 U mg^–1 in cells grown on ASW-AA medium, 0.42 ± 0.05 U mg^–1 in cells grown with pyruvate, and 1.34 ± 0.04 U mg^–1 in cells grown with starch . PGM activity seemed to be induced in the presence of abundant levels of glucose [starch] . Thistendency was also confirmed in nutrient-rich MA-YT medium; wefound that there was a 2.7-fold increase in PGM activity incells grown on starch [0.5%] compared to cells grown on sodiumpyruvate [0.5%] . Using RNA isolated from cells grown in thesemedia, we also performed dot blot experiments and found thattranscription levels were higher in starch-grown cells [datanot shown], which is consistent with the induction of PGM activityobserved in cells grown on starch.

Absence of PGM and PMM activities in the protein products encoded by TK2185 and TK1404. Our biochemical analysis showed that the protein encoded byTK1108 [PGM_Tk] undoubtedly exhibits PGM and PMM activities,which provided strong evidence that the protein represents thetrue PGM/PMM in T . kodakaraensis . It has been shown previouslythat TK1777 encodes a phosphopentomutase with only trace levelsof PGM and PMM activity [13] . In order to determine whetherthe remaining two genes, TK2185 and TK1404, encoded a PGM and/orPMM, we expressed the genes in E . coli and purified the recombinantproteins [Fig . 3] . We found that neither protein exhibited PGMor PMM activity, even when high substrate concentrations [30mM] were used . Altogether, our results revealed that among thefour PMM orthologues in T . kodakaraensis, only one gene actuallyencodes a highly active PGM/PMM.

 
Here, using biochemical analysis, we showed that only one [TK1108]of four putative PMM genes present in the genome of T . kodakaraensis actually encodes a protein with significant PGM activity . This is the first report in which an archaeal PGM gene has been experimentally identified . The protein product also exhibited comparable levels of PMM activity, an activity that also could not be detectedin the other orthologue proteins . During purification from T. kodakaraensis cells, we could not detect any PGM activitiesother than that derived from the TK1108 protein . Although wecannot rule out the possibility that there are other PGMs and/orPMMs that were not expressed under the conditions examined,it is most likely that TK1108 is responsible for both the PGMand PMM activities in T . kodakaraensis.

Another observation that supports our conclusions is that the levels of PGM activity found in T . kodakaraensis cells agreed well with the transcription levels of the PGM_Tk gene . Both levelswere higher in cells grown on starch than in pyruvate-grown cells . This may reflect a role of the enzyme in starch degradation, in which glucose 1-phosphate is produced by the function of starch phosphorylases . Another possibility is that the enzymeis involved in intracellular glycogen synthesis . We also foundputative ADP-glucose synthase [ADP-glucose pyrophosphorylase]genes and a glycogen synthase gene in the T . kodakaraensis genome.When abundant, sugars may be stored in the cells in the formof glycogen.

The phylogenetic tree of PMM genes from Thermococcales [Fig. 2] allowed us to identify the corresponding genes in three Pyrococcusgenomes . TK1108 was closely related to PF0588, PH0923, and PAB0819,suggesting that the latter three genes may encode the PGM/PMMsin their organisms . Interestingly, all three Pyrococcus genesformed gene clusters with the genes encoding a putative mannose-1-phosphateguanylyl transferase, a putative mannosyl 3-phosphoglyceratephosphatase, and mannosyl 3-phosphoglycerate synthase . In particular,the protein products of the latter two genes from P . horikoshiihave been biochemically characterized and have been clearlyshown to exhibit the expected enzyme activities in a biosyntheticpathway for mannoglycerate, a compatible solute [4] . The results obtained in this study, namely, the significant PMM activityof PGM_Tk, agree well with the proposal that genes clustered in the immediate vicinity of the mannosyl 3-phosphoglycerate phosphatase and mannosyl 3-phosphoglycerate synthase genes are involved in mannoglycerate biosynthesis.

Although PGM_Tk displays phosphopentomutase activity [Table 1],it seems unlikely that PGM_Tk is the major phosphopentomutasein T . kodakaraensis . The K_m of the TK1777 product with deoxyribose1-phosphate is slightly lower than that of PGM_Tk, and the k_cat/K_m value is also higher . It has been reported previously that phosphopentomutaseactivity is nearly equivalent in T . kodakaraensis cells grownon pyruvate or starch [13], and this was consistent with thetranscription levels of TK1777 [data not shown] . In contrast,we found here that PGM activity and transcription of TK1108are both upregulated in the presence of starch . With these results,it is reasonable to conclude that TK1108 is the PGM/PMM geneand that TK1777 is the phosphopentomutase gene of T . kodakaraensis.

 
* Corresponding author . Mailing address: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan . Phone: 81-[0]75-383-2777 . Fax: 81-[0]75-383-2778 . E-mail: imanaka@sbchem.kyoto-u.ac.jp.

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What Is Botulism?, What Is Prokaryote?, What Is Genome?, What Is Biotechnology?, What Is Biofilter?, i, Microorganism, s, Microorganisms, r, Bacteria, c, Microbes, a, Microbiology, s, Escherichia coli, e, Escherichia coli, r, Fermentations, c, Clostridia, s, Vancomycin, r, Antibiotic prophylaxis, r, Prokaryotes, s, Minimal inhibiting concentration, i, Staphylococcus, c, Bacillus, i, Escherichia coli, c, Fermentations, n, Escherichia coli, n, Neisseria, i, Neisseria, r, Shigella, a, Escherichia coli, s, S. cerevisiae, e, Escherichia coli, i, Neisseria, o, S. cerevisiae




 

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