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RESEARCH ARTICLE

Purification and Characterization of the 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase From Cymbopogon Flexuosus Leaves

Ashish Kumar Gupta and Deepak Ganjewala*

Amity Institute of Biotechnology, Amity University Uttar Pradesh, Sector 125, Noida-201303, UP, India

*Corresponding Author E-mail: deepakganjawala73@yahoo.com; dganjewala@amity.edu

ABSTRACT:

Cymbopogon flexuosus known as lemongrass is an eminent aromatic grass which produces lemon scented essential oils through the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway. In the second step which is believed to be the first committed step of this pathway, 1-deoxy-D-xylulose-5-phosphate (DXP) simultaneously undergoes an intra-molecular rearrangement and reduction by an enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) to form 2-C-methyl-D-erythritol-4-phosphate (MEP). In the present work we have measured the activities of DXR enzyme accompanying leaf development in C. flexuosus. Also, the DXR enzyme was purified and characterized and referred as CfDXR. The CfDXR activities markedly fluctuated in 1-5^th leaf position of one month old tiller which represents gradient increase in leaf age. The CfDXR activity was recorded maximal in the 1^st and 2^nd leaf position which represents early (immature) developmental stages and declined rapidly in subsequent leaf positions (3^rd-5^th). The CfDXR was purified to homogeneity by three step procedure: ammonium sulfate fractionation, followed by ion-exchange chromatography on DEAE-cellulose and gel exclusion chromatography using sephadex G-75. The purified CfDXR showed a specific activity of 52U/mg protein. It is consists of two identical polypeptides with Mr of 45 KDa as detected by SDS-PAGE. The maximum activity (Vmax) of the purified CfDXR with DXP as substrate was 8.56 μM x min^-1 whereas for NADPH 14.99 μM x min^-1. The purified CfDXR had Km = 3.71 μM for the DXP and 5.99 μM for NADPAH as substrates. The optimum temperature and pH of the CfDXR was 40-60 C and pH 7.5-8.0, respectively. The CfDXR required bivalent cations (Co²⁺, Mn²⁺ and Mg²⁺) for activity. It showed the highest activity in presence of Co²⁺ (1 mM) followed by Mn²⁺ and Mg²⁺. The enzyme when stored at 4 C in 100mM Tris-HCl buffer (pH 7.5) for one month, was quite stable retaining more than 80% of the initial activity.

KEYWORDS: Cymbopogon flexuosus, citral, 1-deoxy-D-xylulose-5-phosphate reductoisomerase, essential oil, 2C-methyl-D-erythritol-4-phosphate pathway

INTRODUCTION:

The genus Cymbopogon comprising of aromatic grasses is one of the most important essential oil producing genus (Khanuja et al., 2005; Ganjewala et al., 2008, Ganjewala, 2009; Ganjewala and Gupta, 2013). Cymbopogon flexuosus commonly known as lemongrass is one of the eminent members in this genus which produce lemon-scented essential oil of high commercial and pharmaceutical importance (Ganjewala, 2009; Ganjewala and Gupta, 2013). The essential oils of C. flexuosus are comprised of monoterpenes mainly the acyclic monoterpene aldehyde citral, which accounts for 80-90% of the total monoterpenes (Ganjewala and Gupta, 2013).

Received on 22.01.2015 Modified on 18.02.2015

Accepted on 08.03.2015 © RJPT All right reserved

Research J. Pharm. and Tech. 8(3): Mar., 2015; Page 320-327

DOI: 10.5958/0974-360X.2015.00053.0

Citral is a recemic mixture of geranial (citral a, E-isomer) and neral (citral b, Z-isomer) that imparts lemon-like aroma to the essential oils of Cymbopogon sp. (Ganjewala and Gupta, 2013). Monoterepenes are C₁₀ compounds belong to the isoprenoids family and derived from geranyl pyrophosphate (GPP). In plants, GPP the universal precursor of monoterpenes is biosynthesized through the head to tail condensation of two C₅ isoprene units called isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP). The IPP in turn is synthesized via the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway (Rohmer et al., 1996; Memelink et al., 2001).

In higher plant the MEP pathway operates in plastidial compartment. The pathway starts with the TPP-dependent condensation of glyceraldehyde-3-phosphate (GAP) and pyruvate to 1-deoxy-D-xylulose-5-phosphate (DXP), which leads to the formation of the IPP and DMAPP in the eight subsequent steps (Rohmer et al., 1996; Lange et al., 1998; Lois et al., 1998). In the second step of the pathway, DXP is simultaneously undergoing isomerization and reduction to form 2C-methyl-D-erythritol-4-phosphate (MEP) by the enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) which requires as cofactor NADPH and bivalent metal ions such as Mn²⁺, Mg²⁺ or Co²⁺ (Kuzuyama et al.,2000; Grolle et al., 2000; Miller et al., 2000). The DXR-catalyzed step is regarded as the first committed step in the MEP pathway that is why the DXR is a key enzyme that regulate and controls biosynthesis of isoprenoids (Veau et al., 2000, Mahmoud and Croteau 2001, Croteau-Paulet et al., 2002). Already, six of the eight enzymes of the MEP pathway have been successfully crystallized and structurally characterized from plants and microorganisms (Proteau, 2004, Cordoba et al., 2009; Quidwai et al., 2014). The genes encoding enzymes of the MEP pathway from many more plants and bacteria have been rapidly cloned and characterized. Detail of the genes encoding enzymes of the MEP pathway from plants have been discussed previously (Ganjewala et al., 2009). The DXR was first cloned and over expressed in E coli which catalyzed the formation of MEP from DXP in a single step in the presence of both NADPH and bivalent cations such as Mg ²⁺, Mn^{2 +} or Co²⁺ (Kuzuyama et al., 2000). Recently, a gene encoding RvDXR from Rauvolfia verticullata was cloned and characterized which is 1804bp long containing a 1425bp open reading frame (ORF), encoding a peptide of 474 AA with molecular mass of 51.3 kDa (Liao et al.,2007). Previously published reports have revealed that there is a positive correlation between the DXR activity and the amount of isoprenoids (chlorophyll and carotenoids) accumulated in transgenic Arabidopsis (Carretero-Paulet et al., 2006) and monoterpenes levels in transgenic peppermint plants (Mahmoud and Croteau, 2001).

Despite the immense pharmaceutical and industrial significance of the Cymbopogon essential oil, till to date very little no gene encoding enzymes of the MEP pathway has been cloned and characterized from the Cymbopogon sp. However, a tremendous progress has been witnessed on cloning and characterization MEP pathway genes from other plant families like lamiaceae, pinaceae, rutaceae and others that provided much deeper insight into biosynthesis and regulation of monoterpenes. The in hand cDNA clone have greatly facilitated the metabolic engineering of the corresponding pathways to improve yield and quality of the essential oils in above plant families. Our recent study in C. flexuosus using radiolabelled substrate, 1-¹³C-glucose and fosmidomycin a potent inhibitor of the DXR followed by quantitative NMR spectroscopy has revealed that in C. flexuosus citral is biosynthesized via the MEP pathway (Fig. 1) (communicated) and not by the classical cytosolic acetate-MVA pathway as was reported previously (Lalitha et al., 1985; Lalitha and Ramasarma, 1986). Here, we report for the first time the DXR enzyme activity during leaf development and its isolation and characterization from C. flexuosus. The DXR enzyme activity markedly fluctuate during leaf development and was correlated with the amount of monoterpenes accumulated, thus indicated its regulatory roles in monoterpene biosynthesis. In future, the gene encoding DXR will be cloned and characterized from C. flexuosus that may provide deeper insight into monoterpene biosynthesis and of use in metabolic engineering of the MEP pathway in Cymbopogon sp. for better yield and quality of essential oils.

Figure 1. MEP pathway of essential oil/monoterpene biosynthesis in Cymbopogon flexuosus. GA-3-P, glyceraldehydes-3-phosphate; DXP, Deoxy-xylulose-5-phosphate; MEP, 2C-methyl-D-erythritol-4-phosphate; IPP, isopentenyl diphosphate; GPP, geranylpyrophosphate; G, geraniol; GA, geranyl acetate; C, citral; EO, essential oil. Enzymes: DXS, 1-Deoxy-xylulose-5-phosphate synthase; CfDXR, Cymbopogon flexuosus 1-Deoxy-xylulose-5-phosphate reductoisomerase; GPPase, geranylpyrophosphatase; GES; geraniol synthase; GAE, geranyl acetate esterase; GDH, geraniol dehydrogenase. TPP, thiamine pyrophosphate; NADPH, nicotinamide adenine dinucleotide phosphate (reduced).

MATERIALS AND METHODS:

Plant material:

Cymbopogon flexuosus cultivar Suvarna plants were grown in the Organic Farm House of the Amity University Uttar Pradesh, Noida, India. The plants were allowed to grow till they are fully expanded (45-50 days). The fully grown tillers were harvested; leaves from tillers were separated according to their position in the tiller from inside to out side. The 1^st (innermost) leaf position represents the youngest leaf stage whereas the 5^th (outermost) leaf stage represented fully matured/expanded stage. In the present study, we used immature (2^nd) leaves of the tiller for isolation of the DXR enzyme.

Chemicals:

Deoxy-xylulose-5-phosphate (DXP), MgCl₂, CoCl₂, MnCl₂, Tris-base, EDTA, 2-mercaptoethanol, ascorbic acid, sucrose, Sephadex G-75, DEAE Cellulose, Polyvinylpyrrolidone (PVPP) and other standard chemicals were purchased from Sigma-Aldrich, Germany

Extraction of DXR:

The DXR was extracted using 50 mM Tris-HCl buffer (pH 7.5) containing sodium metabisulfite (20 mM), 2-mercaptoethanol (10 mM), ascorbic acid (10 mM), sucrose (0.15 M), phenylmethylsulfonyl fluoride (1 mM) and EDTA (1 mM ). Leaf tissues (1 gm) were grounded with extraction buffer (1:4 w/v) in presence of insoluble polyvinylpyrrolidone (PVPP) (50% w/w). The homogenate was squeezed through four layers of muslin cloth and centrifuged at 12,000 × g for 50 min at 4 ºC. The clear transparent supernatant was collected in graduated tubes and used for determination of the DXR activity. Total protein in crude enzymatic extract was quantified by the Bradford method (Bradford, 1976)

DXR assay:

The DXR assay was performed according to previously published report (Mac Sweeney et al., 2005; Grolle et al., 2000; Ramak et al., 2013). The DXR activity was analyzed based on the decrease in A₃₄₀ signal resulted from oxidation of NADPH. The assay mixture was consisted of 50-mM Tris–HCl (pH 7.5) buffer, substrate DXP (1 mM), cofactor NADPH (1 mM) and metal ions (MgCl₂, MnCl₂ and CoCl₂ 2-4 mM) and incubated at 40 ºC for 5 min. The reaction was initiated by adding DXR enzyme to the assay mixture. The oxidation of NADPH was monitored with a UV-VIS spectrophotometer (Shimadzu UV-160) equipped with a cell holder adjusted at 40 °C at 340 nm for 3 min. One unit of DXR activity is defined as the amount of the enzyme that caused oxidation of 1 μmol of NADPH per min. Enzyme activity was expressed as nkatal x min^-1 x mg protein^-1. The molar extinction coefficient values for NADPH at 340 nm is 6220 M^-1cm^-1.

Purification of DXR enzyme:

A three step procedure was used for the purification of DXR enzyme. Unless otherwise indicated, all purification procedures were performed at 4 ºC. In the first step, the enzymic extract prepared from the immature leaves was fractionated by ammonium sulfate ((NH₄)₂SO₄) precipitation as 0-30 %, 30-60 % and 60-90% saturation. Proteins precipitating at all the (NH₄)₂SO₄ saturation steps were collected by centrifugation at 13,000xg for 15 min, resuspended in extraction buffer (50mM Tris-HCl buffer, pH 7.5) and proceed to dialysis. Salting out was done using dialysis bags (Himedia; AV width-42.44nm, AV diameter-25.4 mm, Capacity-5.07 ml/cm) as per molecular weight cut off (MWCO) size of the membrane (25 kDa; 25000 D) against three changes of the same extraction buffer system. The dialyzed sample was applied to activated and pre-equilibrated (Tris-HCl 10 mM, pH 7.5). DEAE cellulose matrix packed in a column (Borosil, 200 x 10 mm column). Elution was monitored spectrophotometrically at 280 nm. The fractions (2 ml each) were collected by washing the matrix with Tris-HCl buffer (10 mM, pH 7.5). Thereafter, the column was eluted by salt step gradient method, using increasing concentration of NaCl (50 mM, 100 mM, 200 mM, 300 mM, 400 mM) in Tris-HCl buffer (10 mM, pH 7.5). Thirty peaks were obtained and fractions corresponding to the peaks were pooled and assayed. Pool 4 from ion exchange chromatography with highest DXR activity was loaded on to pre-washed pre-equilibrated Sephadex G-75 matrix. The column was eluted by isocratic method using Tris-HCl 10 mM, pH 7.5. Twelve fractions of 1 ml each were collected and assayed for DXR activity and protein content. The fractions corresponding to the peaks were pooled and the activity was determined as described above.

Analysis of proteins by SDS-PAGE:

The purified protein was resolved by SDS-PAGE according to Laemmli (1971) using a 12% polyacrylamide gel of 1 mm thickness at constant current of 15 mA. Proteins were visualized by staining of the gel with Coomassie Brilliant Blue R-250. The molecular mass of the protein bands were determined by comparison with the standard molecular marker set (Merck).

Effects of Substrate concentration:

The optimum substrate (DXP) concentration required for the maximum activity of the DXR was determined in terms of V_max and Michaelis constant K_m. The rate (V₀) of the DXR catalyzed reaction was measured using different concentrations of DXP and NADPH ranging from 3 to 15 x 10^-6 moles/L. The V_max and K_m values were determined from the double reciprocal (Line weaver-Burk) plot.

Effects of temperature and Ph:

The temperature and pH optima were determined by performing the enzymatic reaction at different temperatures ranged 0 to100 °C and pH 6 to 9.

Effect of Metal Ions:

Effect of various metals ions (MgCl₂, CoCl₂, MnCl₂, CuCl₂, CaCl₂ and ZnCl₂) were on the activity of the DXR enzyme was evaluated. The enzymatic reaction was performed in presence of each of the metal ions using a similar concentration of 1 mM on DXR enzyme were determined. Effective metals as cofactors MgCl₂, MnCl₂ and CoCl₂ different concentrations were checked for optimum DXR enzyme assay. The DXR enzyme activity was analyzed by as per described.

RESULTS AND DISCUSSION:

Effects of leaf age on DXR activity, Chlorophyll, carotenoid and essential oil content:

To study the developmental changes in the DXR activity, enzymic extracts were prepared from the leaves at 1^st to 5^th position of a one month old C. flexuosus cv. Suvarna tillers. As mentioned above, leaves in a tiller from inside to outside represent a gradient increase in leaf age, the 1^st being the youngest and the outermost fully matured. The results showed that the activity of the DXR expressed as nkatal x mg protein-¹ was maximal in the initial (1-2^nd) leaf positions then rapidly declined thereafter (Table 1). Chlorophylls and carotenoids content (mg/gFW) were maximal in immature leaves (1^st – 3^rd) which decreased thereafter (Table 1). Similarly, the essential oil yield was markedly fluctuated during 1^st -5^th leaf positions with maximum value of yield (mg/10 leaves and %V /FW) during the initial (1^st -3^rd) leaf positions then declined rapidly in the subsequent leaf positions (Table 1). Thus, immature leaves (1^st and 2^nd) are biogenetically most active in the synthesis and accumulation of essential oil. Our previous studies in C. flexuosus using [2-¹⁴C] acetate also revealed that the rate of essential oil biosynthesis was maximal during initial stages of leaf development when leaves are rapidly expanding (Ganjewala et al., 2007a,b; 2008).The higher rate of biosynthesis and accumulation of essential oil could be correlated with higher expression of genes of the MEP pathway that supply precursors for the monoterpene biosynthesis (Vallabhaneni and Wurtzel, 2009; Ruiz-Sola and Rodríguez-Concepción, 2012). The highest activity of DXR observed here in the initial phase of leaf development is most likely to provide precursors IPP/DMAPP for the isoprenoids including monoterpenes (essential oil) biosynthesis. Previous studies on the cloning and over expression of the DXR gene in plants such as spike lavender (Munoz-Bertomeu et al., 2006), Rauvolfia verticillata (Liao et al., 2007), transgenic peppermint (Mamoud and Croteau, 2001) Arabidopsis (Carretero-Paulet et al., 2006) and transplastomic tobacco (Hasunuma et al., 2008) have revealed a correlation between the DXR expression and enhanced essential oil/monoterpene/isoprenoids contents. The expression pattern/level of the DXR enzyme varied with the tissue type and its developmental stages (Liao et al., 2006). Thus the variation seen in the DXR enzyme activity in C. flexuosus is consistent with previous reports. However more detail study is required to confirm the correlation between DXR activity and chlorophyll and carotenoid content.

Table 1. Effects of leaf age on CfDXR activity, essential oil yield, chlorophyll and carotenoids contents in C. flexuosus cv. Suvarna.

Leaf position	CfDXR activity nkatal x mg protein^-1	Essential oil yield		Chlorophyll (mg x FW^-1)			Carotenoids
		mg x 10 leaves^-1	%V /FW	Chl. a (mg x FW^-1)	Chl. B (mg x FW^-1)	Chl. a+b (mg x FW^-1)	(mg x FW^-1)
I	12.96	41.0	0.92	0.50	0.24	0.74	1.69
II	13.09	101.0	1.10	0.47	0.47	0.94	1.62
III	10.34	100.5	0.99	0.50	0.23	0.73	1.61
IV	8.65	61.0	0.59	0.32	0.24	0.56	1.46
V	7.05	52.0	0.55	0.26	0.24	0.50	1.23

Purification and characterization of DXR

The DXR was purified following a three step procedure designed according to the previously published reports. Results of the purification procedure are summarized in Table 2. In the first step, the crude enzyme extract was subjected to precipitation by using ammonium sulfate fractionation followed by dialysis. The results showed that about 60 % of the DXR activity precipitated between 0-30% (NH₄)₂SO₄ saturation with an increase of 1.5 fold purification as compared to the crude extract. Thus obtained enzymatic fraction of DXR was dialyzed using the buffer (50 mM Tris HCl, pH 7.5) which was used for extraction of the enzyme. By the end of this combined procedure, the specific activity of the partially purified DXR measured was 15 nkatal /mg which were relatively higher than 11 nkatal / mg of the crude enzyme. The purified DXR fractions were stored at 4 ºC for the further purification steps. In the second step, the enzyme was purified by ion exchange chromatography on DEAE cellulose column pre-equilibrated with 10 mM Tris-HCl buffer, pH 7.5. The column was eluted by salt step gradient method, using increasing concentrations of NaCl (50-400 mM) in the same buffer. Elution profiles are depicted in Fig. 2. Prominent peaks with maximum DXR activity were recorded in pool 4 comprising of fractions 34-42. The ion exchange chromatography resulted in a significant increase in the specific activity of the DXR which went up to 27.83 nkatal /mg from the previous value of 15 nkatal /mg indicating an almost 2.3 % fold increase in purification fold. In the third or final steps of purification, activated concentrated sample dissolved in 10 mM Tris-HCl buffer was subject to gel filtration chromatography on a Sephadex G-75 column (200 x 10 mm). The column was equilibrated and eluted with the same (Fig. 3). The DXR activity was found in the fractions 6-8. The purified CfDXR showed specific activity 52 nkatal /mg protein with a final 4.8 % fold increases in purification fold. Homogeneity of the DXR containing fraction was evaluated by SDS-PAGE. Single band corresponding to the 45.0 kDa was detected in the gel electrophoretogram which was identified as CfDXR (Fig. 4). The Mr of the CfDXR) has been consisting with the Mr of DXRs reported previously.

Table 2. Summary of purification steps of DXR enzyme of C. flexuosus cv. Suvarna

Purification Steps	Vol. of Fraction (ml)	Protein		Enzyme
		mg x ml^-1	Total (mg)	nkatal x ml^-1	Specific activity (nkatal x mg protein^-1)	Total (nkatal)	Yield (%)	Purification fold
Crude extract	20	1.17	23.52	12.86	10.99	257.2	100	1.00
0-30 % (NH4)2SO4 +dialysis	10	1.05	10.5	15.54	14.80	155.4	60.41	1.34
DEAE-Cellulose	5.8	0.91	5.27	25.33	27.83	146.9	57.12	2.53
Sephadex G-75	2.2	0.59	1.29	30.66	51.96	67.44	26.22	4.72

Figure 2. Ion exchange chromatogram (DEAE-Cellulose column, 200 mm length ×10 mm diameter) of the active fractions received from 0-30 % ammonium sulfate saturation after the dialysis step. The column was equilibrated with 10 mM Tris HCl buffer, pH 7.5, and eluted with a linear NaCl gradient (50 mM-400 mM).	Figure 3. Elution profile of the CfDXR enzyme on Sephadex G-75 (200 mm length ×10 diameter) chromatography. The column was eluted by isocratic method using Tris-HCl 10 mM, pH 7.5.
Figure 4. SDS-PAGE analysis of CfDXR enzyme. Lane 1, protein marker with the indicated molecular masses; lane 2, crude enzyme extract; lane 3, ammonium sulfate precipitated sample; lane 4, DEAE- Cellulose; lane 5, Sephadex G-75 purified CfDXR

The DXRs from microbes and plant are reported to be a homodimer consisted of two unimolecular and identical polypeptides of 42-45 KDa monomers. The DXR catalyzes the conversion of DXP into MEP in presence of the cofactor NADPH and bivalent cations (Mg²⁺, Mn²⁺ or Co²⁺). Here we optimized concentrations of the substrate (DXP), cofactor (NADPH) and metal ions (Mg²⁺, Mn²⁺ or Co²⁺) and determined the kinetic parameters, viz., Km and Vmax for the CfDXR The Km (DXP) and Km (NADPH) determined for the CfDXR were 62.0 and 100.0 nkatal, respectively (Fig. 5 a, b). The pH and temperature optima required for the CfDXR were determined by monitoring the DXR activity at different pH (6-9) and temperature (20-100 ºC). The CfDXR showed highest activity at pH 7.5-8.0 and temperature 40º C (Fig. 5 c, d). Even at 60 ºC CfDXR had 85 % of the maximum activity. The pH and temperature optima determined for the CfDXR are consistent with the results of previously published reports (Kuzuyama et al., 2000). However, the CfDXR is relatively less heat stable than the first characterized E. coli DXR which is reported to be stable even at 80 º C (Kuzyama et al., 2000). The CfDXR has been found to be more similar to DXRs from Zymomonas mobilis reported to be stable at 50-60 º C.

(a)	(b)
(c)	(d)

Figure 5. Effects of (a) substrate DXP; (b) cofactor NADPH concentrations; (c) pH and (d) temperature on CfDXR activity.

The CfDXR also requires bivalent metal for catalyzing the conversion of DXP to MEP. Here we evaluated effects of several bivalent metal ions viz., Co²⁺, Mn²⁺ , Mg²⁺, Cu²⁺ , Fe²⁺ , Ca²⁺ and Zn²⁺ on the activity of the CfDXR. The results showed that these bivalent cations greatly influence the activity of DXR (Fig. 6). Almost a 10 fold increase in the CfDXR activity was recorded in presence of the Co²⁺ (1 mM) followed by Mn²⁺ , Mg²⁺ and Cu²⁺. However the metal ions Fe²⁺, Ca²⁺ and Zn²⁺ did not show any effect on CfDXR activity. Maximal activity of the CfDXR was observed in presence of 2 and 4 mM concentrations of Co²⁺, Mn²⁺ and Mg²⁺. All the previous reports of isolation and characterization of the DXR enzyme bacteria, plants and a malaria parasite (Grolle et al., 2000; Miller et al., 2000; Veau et al., 2000; Altincicek et al., 2000; Yin et al., 2003; Hans et al., 2004) have revealed that they are homodimers of two identical polypeptides of the molecular weights of 42-45 kDa. Further, they require the cofactor NADPH and bivalent metal ions viz., Mn²⁺, Mg²⁺ or Co²⁺ to catalyze the conversion of the substrate DXP into MEP.

Figure 6. Effects of bivalent cations on CfDXR activity. Similar concentration (1 mM) of each metal was used in the enzyme assay.

The kinetic characterization of the DXR has shown that the Km _(DXP) values vary from 2-720 μM depending on the source of enzyme whereas Km _(NADPH) from 0.5 to 190 μM. In the present study, the CfDXR had normal Km values for DXP and NADPH of 3.71 and 5.99 μM , respectively. However, the CfDXR has been found to be quite different from other DXRs in metal ion requirement. The CfDXR preferred Co²⁺ over the other metal ions used. Other characteristic of the CfDXR like stability, pH and temperature optima were almost identical to the previously reported DXRs.

ACKNOWLEDGEMENTS:

I would like to thank Council of Scientific and Industrial Research (CSIR), New Delhi, Government of India for financing our research programme and Senior Research Fellowship to Mr. Ashish Kumar Gupta (Grant No. 1235/EMR-II/2010). I duly acknowledge technical support from AIRF, JNU, New Delhi. Finally, I would like to thank Amity Institute of Biotechnology, Amity University, Noida for providing necessary facilities.

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