Recombinant expression , purification and characterization of Bombyx mori ( Lepidoptera : Bombycidae ) pyridoxal kinase

Pyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme in the metabolism of vitamin B6 (VB6) in Bombyx mori. A fusion expressional vector pET-22b-BPLK-His was constructed using a sub-cloning technique, the recombinant B. mori PLK was then expressed in Escherichia coli, purified and characterized. Bioinformatics were used to deduce the protein structure and genomic organization of this enzyme. Using Ni Sepharose affinity column chromatography, the recombinant protein was purified to very high degree (approximately 90%). The recombinant PLK exhibits a high specific enzymatic activity (1800 nmol/min/mg of protein). The maximum catalytic activity of this enzyme was recorded over a narrow pH range (5.5–6.0) and Zn is the most effective cation for catalysis under saturating substrate concentrations. When only triethanolamine is present as the cation, K is an activator of PLK. A double reciprocal plot of initial velocity suggests that the enzyme catalyses the reaction by means of a sequential catalytic mechanism. Under optimal conditions, the Km value for the substrates of ATP and pyridoxal are 57.9 ± 5.1 and 44.1 ± 3.9 μM. B. mori’s genome contains a single copy of the PLK gene, which is 7.73 kb long and contains five exons and four introns, and is located on the eighth chromosome. The PLK may be a dimer with two identical subunits under native conditions, and it is hypothesized that each monomer contains eight -helices ( 1-8), nine -strands ( 1-9) and two segments of 310 helices. 25 * Corresponding author; e-mail: lqhuang218@yahoo.com.cn to PLP by intracellular PLK (Lumeng et al., 1980; Merrill et al., 1984). In a previous study on VB6 metabolism in B. mori larvae, it was found that dietary PN is absorbed by the midgut, then diffuses into the hemolymph and is actively transported to other organs. PN is first phosphorylated to PNP by PLK, which is then oxidized to PLP by PNPO in every larval organ except hemolymph (Zhang & Huang, 2003). Since B. mori is a large silk-secreting insect, its immense protein turnover needs the timely support of PLP. In order to understand the metabolic mechanism of VB6 in B. mori, the cDNAs encoding PLK and PNPO in B. mori larvae were cloned (Shi et al., 2007; Huang et al., 2009). In this study, the recombinant B. mori PLK was expressed in E. coli as a fusion protein with a hexa-histidine affinity tag, purified by Ni Sepharose affinity column chromatography and characterized. The genomic organization and protein structure of B. mori PLK were also deduced by bioinformatics. MATERIAL AND METHODS


INTRODUCTION
Vitamin B6 (VB6) exists in various forms, pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM) and their phosphorylated derivatives: Pyridoxal 5'-phosphate (PLP), pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP).PLP is the active form of VB6 and acts as an essential, ubiquitous coenzyme in many aspects of amino acid and cellular metabolism.The de novo biosynthesis of VB6 takes place in microorganisms and plants, but animals have lost this ability and it is essential they include it in their diet for the biosynthesis of PLP via a salvage pathway.In the salvage pathway, pyridoxal kinase (PLK) (EC 2.7.1.35)catalyzes the ATP-dependent phosphorylation of PL, PM and PN to form PLP, PMP and PNP, respectively.PNP and PMP are oxidized to form PLP by pyridoxine 5'-phosphate oxidase (PNPO;EC 1.4.3.5).
Like mammals, insects rely on a nutritional source of VB6 to synthesize PLP.When newly moulted larvae of Bombyx mori are reared on a VB6-deficient diet, almost all of them died before moulting to the next instar (Huang et al., 1998).While the biological function of VB6 is similar in all organisms, there are differences between insects and mammals in VB6 metabolism.In mammals, PLP is first synthesized in the liver and then released into the bloodstream in association with albumin.Circulating PLP is dephosphorylated by membrane-associated phosphatase to gain entry into cells and is then converted back to PLP by intracellular PLK (Lumeng et al., 1980;Merrill et al., 1984).In a previous study on VB6 metabolism in B. mori larvae, it was found that dietary PN is absorbed by the midgut, then diffuses into the hemolymph and is actively transported to other organs.PN is first phosphorylated to PNP by PLK, which is then oxidized to PLP by PNPO in every larval organ except hemolymph (Zhang & Huang, 2003).Since B. mori is a large silk-secreting insect, its immense protein turnover needs the timely support of PLP.In order to understand the metabolic mechanism of VB6 in B. mori, the cDNAs encoding PLK and PNPO in B. mori larvae were cloned (Shi et al., 2007;Huang et al., 2009).In this study, the recombinant B. mori PLK was expressed in E. coli as a fusion protein with a hexa-histidine affinity tag, purified by Ni Sepharose affinity column chromatography and characterized.The genomic organization and protein structure of B. mori PLK were also deduced by bioinformatics.

Gene subcloning
Using pET-22b-BPLK as a template, the full coding region of the BPLK-gene was obtained by PCR reaction.The PCR reaction had a total volume of 50 µL, containing 1 µL of templates, 5 µL of 10 × buffer, 200 µM of each dNTP, 300 nM of each primer, 5 U of pfu DNA polymerase and ddH2O 39 µL.To take out the terminator contained in the coding region of BPLK, primers used in the PCR were as follows: sense primer (5'-GGCCATATGTCTCAAGATGATACTCCA-3') and anti-sense primer (5'-GTCTCTCGAGGTTTATTTTCACAGCCTT-3'), which contained Nde I and Xho I recognition sites (underlined).Amplification was performed in a thermal-cycler (Y-Gradient Thermoblock, Biometra, Germany) as follows: 5 min at 94°C; 30 cycles of 30 s at 94°C, 30 s at 55°C and 60 s at 72°C, followed by 10 min at 72°C.The PCR product was purified using DNA Gel Extraction kit, and digested with Nde I and Xho I restriction enzymes.The digested fragment was then purified again and ligated with T4 DNA ligase into pET-22b(+), previously digested with the same enzymes.The resulting recombinant plasmid was named pET-22b-BPLK-His, which contained a T7 promoter, B. mori PLK gene fused to a C-terminal hexahistidine affinity tag sequence and T7 terminator.
The plasmid was used to transform the competent cells of E. coli DH5 for sequencing.The sequencing study was accomplished by Sangon Bio-technology Company (Shanghai, China) using the dye terminator method and an ABI 3730 automatic DNA sequencer.

Protein expression and purification
After the electrophoretic analysis and sequencing analysis of the DNA fragment, pET-22b-BPLK-His was used to transform E. coli Rosetta (DE3) cells for protein expression.The single bacterial colony of E. coli Rosetta (DE3), harbouring pET-22b-BPLK-His, was cultured in 20 mL Luria-Bertani (LB) medium with ampicillin (50 µg/mL) and chloramphenicol (3.4 µg/mL) on a shaker platform, overnight, at 37°C, and 4 mL of the overnight cultures was then inoculated into 400 mL LB medium with ampicillin (50 µg/mL) and chloramphenicol (3.4 µg/mL).The inoculum was grown at 37°C with vigorous shaking to an OD600, approximately 0.4-0.6.IPTG was added to a final concentration of 1.0 mM and the cells further incubated and shaken for 12 h at 16°C.Cells were harvested by centrifugation (6,347 × g at 4°C for 15 min), re-suspended in cold 1 × phosphatebuffered saline (PBS) (10 mM sodium phosphate, pH 7.4, 140 mM sodium chloride, 5 mM potassium chloride) and collected by centrifugation (6,347 × g at 4°C for 15 min).
The pellets were re-suspended in loading buffer (20 mM sodium phosphate, pH 7.4, 20 mM imidazole, 0.5 M NaCl).Protease inhibitor cocktails were added according to the manufacturer's instruction.Unless otherwise specified, all subsequent steps were performed at 4°C.The suspensions were then broken up by sonication in ice and centrifuged at 12,840 × g for 10 min.
For the purification, about 10 mL of the supernatant was loaded onto a chromatography column (24 mL) filled with 3 mL of Ni Sepharose and washed extensively with sodium phosphate buffer with increased imidazole concentration (= 150 mM) to remove un-specifically bound proteins, including E. coli PLK.Subsequently the histidine-tagged protein was eluted in 16 mL of sodium phosphate buffer containing 300 mM imidazole.The purified B. mori PLK was concentrated to about 0.42 mg/mL with a Millipore (10,000 Da cut-off) in a buffer of 70 mM potassium phosphate, pH 6.5.The above description is of a typical run.
The purity and homogeneity of the fractions and the subunit molecular weight of the PLK were estimated by 1-dimensional SDS-PAGE.Protein concentrations were determined by the method of Bradford using bovine serum albumin as standard.The molecular weight was determined by injecting approximately 3 mg purified B. mori PLK into a Sephadex G-100 column (1 × 100 cm).Samples of lysozyme, chymotrypsinogen, ovalbumin and hemoglobin were used as markers of known molecular weight.The elution buffer was 50 mM Tris-HCl (pH 7.5) and the flow rate 0.4 ml/min.

Assay of PLK activity
Using PL as a substrate PLK activity was determined using the method of Sakurai (Sakurai et al., 1993) with some modifications.The formed product, PLP was determined by reaction with phenylhydrazine to enhance the sensitivity.Unless otherwise specified, all activity assays were performed three times.Purified enzyme (about 12 µg) was incubated at 37°C for 10 min in an assay buffer of 70 mM potassium phosphate (pH 5.5) with 1.0 mM PL, 1.0 mM ATP and 0.5 mM ZnCl2 in a total volume of 1 mL.The reaction was stopped by adding 100 µL of 100% (w/v) trichloroacetic acid, centrifuged at 11,400 × g for 10 min and any precipitate that formed discarded.One hundred micro liters of phenylhydrazine in 10 M sulphuric acid was added to the mixture of 1 mL of supernatant and 2 mL of assay buffer.Enzyme activity was determined using a spectrophotometer at 410 nm for the first 2.5 min (Unico UV-2600 spectrophotometer, Unico instrument Co., Ltd, shanghai, China).The blank was prepared by adding ATP after stopping the reaction with trichloroacetic acid.A unit of activity is defined as the nmol of PLP formed per minute per mg of protein at 37°C.A standard curve of the absorbance at 410 nm against different concentrations of PLP standard was constructed for calculating the PLP generated in the enzyme assay.
The pH dependence of the PLK activity was measured between pH 4 and 9, with an assay buffer of 70 mM citric acid/potassium orthophosphate/boric acid solutions adjusted with NaOH.Activity assays were performed at various pHs at 37°C for 10 min.The effect of temperature on the PLK activity and stability were determined at temperatures from 20 to 55°C.Activity was measured for 10 min at different temperatures.For stability assays, the enzyme was pre-incubated for 1 h at the indicated temperature, and residual activity was then assayed at 37°C as described above.Effect of divalent cations on the activity of the PLK was measured in the standard assay condition with 0.5 mM of various divalent cations instead of ZnCl2.
To compute the enzyme kinetic data, about 10 µg of the purified enzyme was used in each assay, and the concentrations of substrates were varied in the range 2-800 µM.At this enzyme concentration, the initial enzymatic rate was linear.Using PL as the variable substrate and ATP as the fixed substrate, the enzyme kinetic mechanism was estimated from double reciprocal plots.

Analysis of genomic organization and protein structure of the enzyme
The B. mori PLK cDNA (GenBank accession number: DQ452397) was used as the query to search the genomic database of B. mori (http://www.silkdb.org/,http://sgp.dna.affric.go.jp/) for the gene, and the gene structure was analyzed using software SeqVISTA (http://www.bio-soft.net/format.html).
The secondary structure of the purified PLK was quantitatively determined by circular dichroism (CD) spectrum analysis.Determination of CD spectrum was carried out on a JASCO-J810 spectropolarimeter (Jasco Corporation, Japan).The instrument conditions were as follows: measurement range, 260-190 nm; data pitch, 0.2 nm; data points, 351; band width, 3 nm; response, 1 s; sensitivity, standard; scanning speed, 100 nm/min; accumulation, 3; cell length, 0.1 cm; temperature, room temperature; control and analysis software, Spectra Managermr.The purified PLK was dissolved in 50 mM potassium phosphate (pH 7.4) buffer with a concentration of 0.1 mg/mL.
The three-dimensional structure of B. mori PLK monomer was predicted by the method of homology modelling, using the human PLK (PDB accession number: 2YXT) as a template.Amino acid sequence was submitted to SWISS-MODEL server (http://www.swissmodol.expasy.crg/)for homology modelling.The result was analyzed by visual software Pymol ( http://www.bio-soft.net/3d/pymol.htm).The location and mode of substrate binding in B. mori PLK was deduced by analogy with the known crystal structures of mammalian PLK in complex with substrates.

Recombinant expression and purification of B. mori PLK
With the help of colony PCR, about a 900 bp product was obtained from fusion expressional vector pET-22b-BPLK-His (Fig. 1   Using the polyhistidine as a fusion tag, the over expressed protein was found in the soluble fraction.Using Ni Sepharose affinity column chromatography, the PLK was purified to over 90% homogeneity judged from 1-dimensional SDS-PAGE analysis (Fig. 2).Table 1 summarizes the purification of PLK.The enzyme was purified about 26-fold, and the final yield of the enzyme was 82% of the homogenate activity.
The dimeric PLK molecular mass was determined to be 68 kDa by Sephadex G-100 gel filtration (Fig. 3).On a reducing SDS-PAGE gel (Fig. 2), the PLK, including one 6 × histidine tag in the C-terminus, appeared as a single band of about 33.9 kDa computed by software of Quantity one (http://www.seekbio.com/soft/275.html) based on the protein marker.The data suggest that the recombinant PLK is a dimer with two identical subunits under native conditions.

Catalytic properties of recombinant B. mori PLK
Fig. 4 shows the effect of pH on the catalytic activity of the purified PLK.The PLK has maximum catalytic activity in the narrow pH range of 5.5-6.0 and with Zn 2+ , and the enzyme was inactive below pH 4.5.Enzyme activity decreased slowly above pH 6.0, to approximately  35% at pH 8.5.Performing the pH dependent assay with Mg 2+ , the optimum pH for the PLK activity was 6.0.The enzyme displayed optimal activity at 50°C, and its greatest stability was below 40°C (data not shown).At pH 5.5 and 37°C, the time course of enzyme activity was linear for up to 40 min (data not shown).
Figs 5 and 6 show the effect of divalent cation on PLK activity.Under saturating PL and ATP concentrations, Zn 2+ is the most efficient cation for catalysis, analyzed with phosphate buffer (pH 5.5) or triethanolamine buffer (pH 7.3).At about a 1 µM substrate concentration, Mg 2+ , however, stimulates the activity (Fig. 6).Enzyme activity was also measured using a monovalent cation at saturating PL and ATP concentrations, which revealed that K + is an activator of PLK when only triethanolamine is present as the cation (Fig. 7).If instead of the phosphate buffer an acetate buffer is used, enzymatic activity was reduced to 74% and almost no activity was recorded when a citrate buffer was used (Fig. 8).
An initial velocity study using PL as the variable substrate and ATP as the fixed substrate gave a family of lines intersecting to the left of the vertical axis (Fig. 9), which eliminated the possibility of a ping-pong catalytic mechanism.Such an intersecting pattern suggests that the enzyme catalyzes the reaction by means of a sequential catalytic mechanism.As the point of intersection of the lines is below the horizontal axis, it reveals that the com-bination of fixed substrate and enzyme affects the Km value of the variable substrate, which increases with increase in the concentration of the fixed substrate.Under optimal conditions, the Km values of PLK for ATP and PL were determined as 57.9 ± 5.1 and 44.1 ± 3.9 µM.Table 2 summarizes the kinetic parameters of PLK including the Km, Vmax, kcat and kcat/Km.

Genomic organization of B. mori PLK
Using cloned B. mori PLK cDNA as a query to search against the B. mori genomic database (http://www.silkdb.org/), the PLK gene was located at nscaf2828:3863864-3871593 with gene number BGIBMGA005472-TA.From the other B. mori genomic database (http://sgp.dna.affric.go.jp/), the PLK gene was located at chr8:11034281-11042010Bm_scaf19:3870164-3877893 with gene number BGIBMGA005472.The two genomic databases gave the same result.B. mori contains a single copy of the PLK gene on the eighth chromosome and no other homologous genes were found.The PLK gene spans a region about 7.73 kb long, and contains five exons and four introns (Fig. 10).Within the region from -26 to -68, relative to the transcription start site (-CCATAT-), typical TATA-like and CAAT-like boxes are identified.All exon/intron boundaries contain the canonical 5' donor GT and 3' acceptor AG sequences.At the 3' region of the PLK gene, some A-tailing sequences were identified.
Fig. 12 shows the amino acid sequence alignment of B. mori, human, sheep, A. thaliana, wheat and E. coli PLKs.The B. mori PLK contains 298 amino acid residues with a theoretical molecular mass of 33.1 kDa and pI value of about 6.3.The amino acid sequence shares a 50% identity with that of human PLK, and 48% with sheep, 46.7% with A. thaliana, 44.48% with wheat and 32% with E. coli, respectively.B. mori PLK contains conserved amino acid sequence motifs that may be involved in substrate binding or catalysis of the PLK family.A distinctive characteristic of B. mori PLK is that its sequence is 10 or more residues shorter than that of PLK from mammals and plants.Compared with B. mori PLK, the sequence exhibits extension of the N-terminal in plants, and increase in nonconservative residues in mammal.Secondly, the key peptide loop, which is thought to play a significant role in the functioning of PLK (Safo et al., 2006), consists of eight residues in B. mori PLK and 12 in mammals.
Fig. 13 (B) shows the monomer structure of B. mori PLK, predicted using homology modelling, with the help of software Pymol (http://www.bio-soft.net/3d/pymol.htm).Each monomer consists of eight -helices ( 1-8), nine -strands ( 1-9) and two segments of 310 helices.The 1-8 -strands constitute a central contorted -sheet flanked by 2, 3, 4, 5 and 6 on one side, and 1, 7 and 8 on the other side.The overall folding pattern is a three-layer sandwich, which is common in the ribokinase super-family.(Li et al., 2002(Li et al., , 2004)).On the enzyme surface, there is a cavity with a negative charge located along one edge of the central -sheet and this high negative charge attracts substrates with a positive charge, such as the pyridine ring of VB6 and the adenine ring of ATP, which bind there.The ATP-binding site is positioned in a shallow groove formed by the hydrophobic side chains of surrounding residues.Residues that interact with the phosphate groups of ATP are Ser 185 , Thr 146 , Asp 115 , Asn 148 , Asp 120 , Gly 220 , Tyr 125 and Thr 219 ; and residues Leu 197 , Lys 211 , Phe 216 , Ser 199 , Ile 209 and Leu 249 interact with the adenine ring of ATP.The PL-binding site is located in a pocket in the opposite direction of ATP, and consists of Tyr 86 , Val 21 , Thr 217 , Ser 14 , Thr 49 and Asp 221 .

DISCUSSION
A large number of genes/cDNAs encoding PLK have been isolated from mammals, microorganisms and plants, and their sequences submitted to the GenBank.A sequence homology search using the Dnaman program found several close homologs of PLK, from both prokaryotes and eukaryotes.The sequence identity with the human enzyme ranges from 24% to 90%.The amino acid sequence of B. mori PLK shares a 50% identity with that of human PLK.
In mammals, PLK is a dimer of identical subunits, each with an estimated molecular mass of approximately 35 kDa.The dimer can dissociate reversibly into catalytically active monomers (Kwok et al., 1987).As in mammals, the B. mori PLK also may be a dimer with two identical subunits under native conditions.The monomer molecular mass of B. mori PLK, however, is 33.1 KDa and smaller than the mammalian counterpart.
Previous structural analysis of sheep brain PLK complexes reveals that there is a key 12-residue loop (Gly-117 to Val-128) over the active site, which has an important role in the catalytic process.After ATP binding, the loop partially covers the ATP-binding site and prevents the unproductive hydrolysis of ATP; when substrates are absent, the loop exhibits a different conformation and occupies neither the ATP-binding site nor the PL-binding site (Li et al., 2002(Li et al., , 2004;;Tang et al., 2005).The conformation of the corresponding segment in human PLK is not a loop, but a -strand/loop/ -strand flap (Cao et al., 2006).A multiple sequence alignment of PLKs from different species (Fig. 12) indicates that the key peptides in B. mori consist of eight residues, similar to that in plants and E. coli.It is suggested that the length and conformation of the peptide might serve as an indicator of where it is along the evolutionary pathway of the PLK family from simple to complex.
The structure of the PLK active site is well studied in mammals (Li et al., 2002(Li et al., , 2004;;Cao et al., 2006).Shared by human and sheep, the residues that interact with the phosphate groups of ATP are Ser  (Cao et al., 2006).In B. mori PLK, the residues are replaced by Gly, Ile and Leu, respectively.It is suggested that like human PLK, B. mori PLK has more affinity with ATP than sheep PLK.In addition, Asn 121 , which has a positive charge over the ATP-binding site in sheep brain PLK, is replaced by negative charged residue Asp 121 , and the residue Arg 120 in the key peptide loop of sheep brain PLK is replaced by Trp 120 in human PLK (Cao et al., 2006).Two amino acid residue positions are found to be absent from B. mori PLK.Moreover, residue Asn 45 in human PLK is conserved among all species aligned in this research, but is substituted by Thr in B. mori PLK.A key residue His 59 in E. coli PLK, interacts with the aldehyde group at C-4 of PL and may also determine if residues from the key peptide loop can fill the active site in the absence of the substrate (Safo et al., 2006).The residue His is substituted by Ala in B. mori PLK.
In E. coli, the metal ion Mg 2+ and K + are required for enzyme activity (Li et al., 2002;Safo et al., 2006).In contrast, Zn 2+ and K + have been proposed to be the metal ions needed for the activity of both human PL and sheep PL kinases (Li et al., 2002).However, a more recent study of the human enzyme showed that at non-physiological concentrations of the substrate and/or at pH 6, at which the previous assays were performed, Zn 2+ does stimulate activity (McCormick et al., 1961;White & Dempsey, 1970), but under physiological conditions at pH 7.3, Mg 2+ is the required divalent metal ion and Zn 2+ inhibits the reaction (Di Salvo et al., 2004).At saturating PL and ATP concentrations, both Na + and K + activate human PLK, with Na + resulting in a six-fold increase in activity and K + only a 2.5-fold increase (Musayev et al., 2007).When the activity of the recombinant B. mori PLK using monovalent cations under saturating PL and ATP concentrations was measured it was found that K + is also an activator of the enzyme, whereas Na + did not activate PLK.The pH dependent study with Mg 2+ and Zn 2+ showed optimum enzyme activity at pH 6.0 and 5.5, respectively, with the Zn enzyme exhibiting more activity.At pH 7.3 with triethanolamine buffer, Zn 2+ was still the most effective divalent cation for the catalysis of B. mori PLK.This data suggest that the optimal activity of B. mori PLK is recorded in acidic environments.Under physiological substrate concentrations, Mg 2+ slightly stimulated the activity of B. mori PLK.It was not possible to detect inhibition by Zn 2+ because the substrate concentrations were so low that the initial velocity could not be measured accurately.The mechanism of phosphorylation has been elucidated for sheep and E. coli enzymes, and follows a random sequential substrate addition (Li et al., 2002(Li et al., , 2004;;Safo et al., 2004Safo et al., , 2006)).Using PL as the variable substrate and ATP as the fixed substrate, a double reciprocal plot of initial velocity also suggests a sequential catalytic mechanism for the B. mori PLK.
Table 4 summarizes the genomic organization of PLKs from B. mori, human, A. thaliana, malarial parasite and E. coli.The human PLK gene and that of A. thaliana contain more than ten exons, and that of B. mori and the malarial parasite five and three exons, respectively.The B. mori PLK gene has shorter introns than the human PLK gene.These differences may be related to the evolution of the PLK family and the complexity of the regulation of PLK gene expression.
In conclusion, the results of recombinant expression, purification and characterization of B. mori PLK are presented.This is the first report on the characterization of a PLK in insects.B. mori PLK contains signatureconserved amino acid sequence motifs of the PLK family.The catalytic properties and protein structure of B. mori PLK are similar to those of human PLK in terms of mass, but some distinguishing feature of the PLK was also observed in this study.

Fig. 1 .
Fig. 1.Electrophoresis of the products of colony PCR.Lane M is DNA marker; Lane 1 is BPLK; Lane 2 is negative control.

Fig. 4 .
Fig. 4. Effect of pH on the catalytic activity of the recombinant B. mori PLK.Filled square -pH dependent assay with Zn 2+ , and open square -pH dependent assay with Mg 2+ .Fig. 6.Effect of Zn 2+ and Mg 2+ at different physiological substrate concentrations on the activity of the recombinant B. mori PLK.Filled square -Zn 2+ ; open square -Mg 2+ .

Fig. 5 .
Fig. 5. Effect of divalent cation on the activity of the recombinant B. mori PLK.Filled column -activity assay with phosphate buffer (pH 5.5); open column -activity assay with triethanolamine buffer (pH 7.3).

Fig. 7 .
Fig. 7. Activity of B. mori PLK in the presence of either K + or Na + .Monovalent cation was added in increasing concentrations to the PLK in triethanolamine buffer (pH 7.3).Filled square -K + ; open square -Na + .Fig. 8. Assays of B. mori PLK activity using 70 mM, pH 5.5 sodium phosphate buffer (A), sodium acetate buffer (B) and sodium citrate buffer (C).

Fig. 13
Fig.13 (C) shows the location and mode of substrate binding in B. mori PLK deduced by analogy with the structures of sheep brain PLK in complex with various substrates(Li et al., 2002(Li et al., , 2004)).On the enzyme surface, there is a cavity with a negative charge located along one edge of the central -sheet and this high negative charge attracts substrates with a positive charge, such as the pyridine ring of VB6 and the adenine ring of ATP, which bind there.The ATP-binding site is positioned in a shallow groove formed by the hydrophobic side chains of surrounding residues.Residues that interact with the phosphate groups of ATP are Ser 185 , Thr 146 , Asp 115 , Asn 148 , Asp 120 , Gly 220 , Tyr 125 and Thr 219 ; and residues Leu 197 , Lys 211 , Phe 216 , Ser 199 , Ile 209 and Leu 249 interact with the adenine ring of ATP.The PL-binding site is located in a

Fig. 12 .
Fig. 12. Sequence alignment of PLKs from B. mori, humans, sheep, A. thaliana, wheat and E. coli.Dots for identical residues, dashes gaps introduced to maximize similarity.The key peptides of loop are marked by filled triangles.ATP-binding site and PLbinding site are boxed, and ATP-binding site is coloured grey.
187 , Thr 148 , Asp 113 , Asn 150 , Asp 118 , Gly 234 , Tyr 127 and Thr 233 .The residue Tyr 84 is on one side of the pyridine ring of PL and makes a -interaction with the pyridine ring, whereas Val 231 and Val 19 are on the other side, interacting with PL by a hydrophobic effect.The N-1, O-3 and O-5 atoms of PL form hydrogen bonds and hydrophobic interact with the side chains of Ser 12 , Thr 47 and Asp 235 , respectively.Residues Val 41 , Phe 43 , Val 14 , Val 56 , Trp 52 and Val 115 all contribute to forming a hydrophobic environment for the binding of PL to the active site; especially Tyr 84 , Asp 235 and Ser 12 , share the function of determining substrate specificity.All of these important residues are found to be conserved in B. mori PLK, except Val 231 and Trp 52 .The residue Val is replaced by Thr in plants and B.mori PLK, whereas Trp is replaced by Ile in B. mori PLK.Residues Ala 201 , Met 223 and Met 263 , which interact with the adenine ring of ATP in sheep brain PLK through hydrophobic interactions, are substituted, respectively, in human PLK by more hydrophobic amino acids Val 201 , Ile 223 and Leu 263

Fig. 13
Fig. 13.A -Monomer structure of human PLK obtained from PDB database with the help of visual software Pymol.B -Monomer structure of B. mori PLK predicted by homology modelling.C -Monomer structure of B. mori PLK with active site.The ATP phosphate group-binding site, ATP adenine ring-binding site and PL-binding site are coloured yellow, red and purple, respectively.

TABLE 1 .
).DNA sequencing demonstrated that the PCR product had a B. mori PLK gene and a C-terminal hexa-histidine tag sequence.Summary of the purification of recombinant B. mori PLK.