SciELO - Scientific Electronic Library Online

vol.12 número3Establishment of an efficient callus induction and plant regeneration system in Pakistani wheat (Triticum aestivum) cultivarsGroup III PLA2 from the scorpion, Mesobuthus tamulus: cloning and recombinant expression in E. coli índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados




Links relacionados

  • En proceso de indezaciónCitado por Google
  • No hay articulos similaresSimilares en SciELO
  • En proceso de indezaciónSimilares en Google


Electronic Journal of Biotechnology

versión On-line ISSN 0717-3458

Electron. J. Biotechnol. v.12 n.3 Valparaíso jul. 2009 

Molecular Biology and Genetics

Microbial Biotechnology

Electronic Journal of Biotechnology ISSN: 0717-3458 Vol. 12 No. 3, Issue of July 15, 2009
© 2009 by Pontificia Universidad Católica de Valparaíso -- Chile Received September 2, 2008 / Accepted April 29, 2009
DOI: 10.2225/vol12-issue3-fulltext-14 How to reference this article

Gene expression and characterization of 2-keto-3-deoxy-gluconate kinase, a key enzyme in the modified Entner-Doudoroff pathway of Serratia marcescens KCTC 2172

Yong-Seok Lee
Department of Biotechnology
Faculty of Natural Resources and Life Science
Dong-A University
Busan, 604-714, Korea

In-Hye Park
Department of Biotechnology
Faculty of Natural Resources and Life Science
Dong-A University
Busan, 604-714, Korea

Ju-Soon Yoo
Department of Food Science and Nutrition
Dong-Ju College
Busan, Korea

Hae-Sun Kim
Department of Biotechnology
Dong-A University
Busan, 604-714, Korea

Soo-Yeol Chung
Department of Food Science
Dong-Ju College
Busan, 604-715, Korea

Muni Ramanna GariSubhosh Chandra
Department of Biotechnology
Dong-A University
Busan, 604-714, Korea

Yong-Lark Choi*
Department of Biotechnology
Faculty of Natural Resources and Life Science
Dong-A University 840, Hadan-dong
Saha-gu, Busan, 604-714, Korea
Tel: 82 51 200 6536
Fax: 82 51 200 6536

*Corresponding author

Financial support: Research fund of Dong-A University.

Keywords: 2-keto-3-deoxygluconate kinase, carbohydrate kinase, purification, Serratia marcescens KCTC 2172.


GST: Glutatione S-transferase
KDG: 2-keto-3-deoxygluconate
KDGK: 2-keto-3-deoxy-gluconate kinase
KDPG: 2-keto-3-deoxy-6-phophogluconate
KG: 2-ketogluconate
mms: maltose metabolism stimulation
ORF: open reading frames
SDS-PAGE: sodium dodecyl sulphate-spolyacrylamide gel electrophoresis

Abstract   Reprint (PDF)

We cloned 2-keto-3-deoxy-gluconate kinase (KDGK), which catalyzes the phosphorylation of 2-keto-3-deoxygluconate (KDG) to 2-keto-3-deoxy-6-phophogluconate (KDPG) from Serratia marcescens KCTC 2172. The nucleotide sequence revealed a single open reading frame containing 1,208 bp and encoding for 309 amino acids, with a molecular weight of 33,993 Da. The enzyme was purified via GST affinity chromatography. The putative KdgT binding site was detected upstream of the initial codon. The KDG kinase utilized 2-ketogluconate (KG) and KDG as substrates. The optimal temperature and pH for KDGK activity were 50ºC and 8.0, respectively.

Materials and Methods

  • Cloning and expression of the kdgK gene
  • Purification of recombinant KDGK
  • Enzyme assay
  • Polyacrylamide gel electrophoresis
  • Effect of temperature and pH on enzyme activity
  • Cloning and expression of the kdgK gene
  • Alignment of the KDGK operator with the consensus of the KdgR binding site
  • Amino acid sequence similarities between KDG kinase and other proteins
  • Purification of the recombinant enzyme
  • Effect of temperature and pH on KDG kinase activity

    Figure 1
    Figure 2
    Figure 3
    Figure 4
    Figure 5
    Figure 6
    Figure 7
    Table 1
  • Serratia is gram-negative bacteria, classified in the large family of Enterobacteriaceae. Serratia can be distinguished from other genera by its production of three special enzymes DNase, lipase and gelatinase. Another characteristic feature of the Serratia among the family Enterobacteriaceae, in the tribe Klebsiella has been widely considered a red-pigmented organism. Serratia, like other Enterobacteria, grow well on ordinary media under anaerobic and aerobic conditions. They grow well on synthetic media using various carbon sources (Giri et al. 2004). Serratia marcescens is an enteric bacterium closely related to Escherichia coli and secretes several enzymes through the membrane into culture broth, including nucleases, lipases, proteases, chitinases (Gal et al. 1998), coenzyme A transferase, cyclic AMP receptor protein, acetoacetyl coenzyme A transferase (Yoo et al. 2000; Yoo et al. 2001), maltose metabolism stimulation, poly-phosphate kinase and exopolyphosphatase (Lee et al. 2000; Lee et al. 2006).

    2-Keto-3-deoxygluconate kinase (KDGK), which catalyzes the phosphorylation of 2-keto-3-deoxy-gluconate (KDG), plays a role in the carbohydrate metabolism of Clostridia and some of the aerobic bacteria (Ohshima et al. 2007). KDG is an important intermediate of the degradation of extracellular pectin, which is processed and incorporated into cells, and then converted into KDG in Erwinia chrysanthemi (Hugouvieux-Cotte-Pattat et al. 1996). In E. coli (Mandrand-Berthelot et al. 1984) and E. chrysanthemi, extracellular KDG is also utilized by assimilation via the KDG transporter. KDG is also reported to be produced from D-glucosaminate by D-glucosaminate dehydratase in Pseudomonas fluorescens (Iwamoto and Imanaga, 1991).

    The pathways of pectin and galacturonate catabolism in bacteria converge to form a common intermediate, KDG, which is phosphorylated by KDG kinase. The KDGK product is the last step of pectin catabolism that is identified at the molecular level in E. chrysanthemi. In this organism, breakdown of pectic polymers involves various types of pectinolytic enzymes. This cleavage gives two kinds of monomeric sugar: 5-keto-4-deoxyuronate (KDU) and D-galacturonate. These monomers are catabolized by two independent pathways converging through a common intermediate: KDG. The ketodeoxyuronate catabolic pathway is composed of successive isomerization and oxidoreduction steps catalyzed by the kduI and kduD gene products (Condemine et al. 1986; Condemine and Robert-Baudouy, 1991). The galacturonate catabolic pathway consists of three enzymes encoded by the operon uxaCBA. Similarly, hexuronate, glucuronate, can be degraded by three parallel steps catalyzed by the uxaC, uxuB, and uxuA gene products (Hugouvieux-Cotte-Pattat and Robert-Baudouy, 1987). The common catabolic product KDG, is then phosphorylated by the KDGK, yielding 2-keto-3-deoxy-6-phophogluconate (KDPG), which is cleaved by the KDG aldolase gene product to give intermediates of the general cellular metabolism (Hugouvieux-Cotte-Pattat and Robert-Baudouy, 1985). In order to find out whether the increased expression of KDGK under the direction of a strong promoter such as tac promoter is necessary and genes for the putative S. marcescens 2-keto-3-deoxygluconate kinase are introduced into a bacterial expression vector pKK223-4. The aim of the present study was to express pKKDGK in Escherichia coli and determine the enzymological properties of the purified product to obtain a better understanding of the modified ED pathway in Serratia marcescens KCTC 2172.

    Materials and Methods

    Cloning and expression of the kdgK gene

    In a previous study (Yoo et al. 2000; Yoo et al. 2001), we constructed several clones from a genome library of Serratia marcescens KCTC 2172. In this study, pCKB17, one of the clones from the genome library, was analyzed in more detail. The nucleotide sequence of pCKB17 was determined, and a restriction map of the cloned DNA fragment and several recombinant subclones was constructed (Figure 1). The open reading frame of the cloned kdgK gene was amplified via PCR with the 5’ and 3’ primers: 5’-AGCCCGGGCTTATTGA-CGATGACTATC-3’ and 5’-CATAATCAATAAGGCGGC-GGCGAAGCTTCC-3`, harboring the SmaI and HindIII restriction enzyme sites. pCKB17 was utilized as a template DNA. Following digestion with SmaI and HindIII, this PCR product was cloned into pKK223-4 and digested with the corresponding enzymes for the construction of pKKDGK. The culture was cultivated in M9 minimal medium containing 0.1% glucose and casamino acids (Le et al. 2006). E. coli BL21 (DE3) harboring pKKDGK was induced for over-expression with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at the mid-exponential growth phase, then incubated for 3 hrs at 37ºC.

    Purification of recombinant KDGK

    Cells were harvested via centrifugation (6000 rpm for 15 min at 4ºC), washed with buffer 1 x PBS buffer (Diluted 10 x PBS, 1.4 M NaCl, 27 mM KCl, 100 mM Na2HPO4 and 18 mM KH2PO4 at pH 7.3) and re-suspended in 1 x PBS buffer. The cells were disrupted by sonication and the supernatant was acquired through 30 min of centrifugation at 13000 rpm at 4ºC. The sonicated solution was then loaded onto a GSTrap FF column (Amersham Pharmacia Biotech), equilibrated with 1 x PBS, and eluted with 10 mM reduced glutathione in 50 mM Tris-HCl (pH 7.5) at a flow rate of 1 ml/min. The eluted fractions were dialyzed overnight against PreScission cleavage buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA and 1 mM dichlorodiphenyltrichloroethane at pH 7.0) and concentrated using Amicon Ultra-4 strips (Millipore, Bedford, MA, USA). The purified fusion protein was then digested with PreScission protease for 12 hrs at 5ºC to remove the GST region, and then the digested fusion protein was loaded onto a GSTrap FF column chromatography as described above. The unbound fraction was collected and employed as the purified enzyme.

    Enzyme assay

    The enzyme coupling assay for KDGK was conducted by the method described by Ohshima et al. (2007). The standard reaction system consisted of 50 mM Tris-HCl (pH 8.5), 10 mM KDG, 10 mM glucose, 2.5 mM ATP, 10 mM MgCl2, 6H2O, 1 U of yeast glucose-6-phosphate dehydrogenase, 1 U of ADP-glucokinase, 1 mM NADP, and the enzyme in a total volume of 1.0 ml. The reaction was initiated via the addition of ATP, and an increase of absorbance at 340 nm, which is reflective of NADPH formation, followed at 50ºC. One unit of enzyme activity was defined as the amount of enzyme required to generate 1 µ mol of ADP per min under the standard assay conditions, and the specific activity was expressed in U/mg of protein. Protein concentrations were determined using the method of Bradford (1976), with bovine serum albumin as the standard. ADP-dependent glucokinase was acquired from Asahi Pharma, Tokyo.

    Polyacrylamide gel electrophoresis

    Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted by the method of Laemmli (1970). After SDS-PAGE, the gel was stained with 0.05% Coomassie brilliant blue R-250.

    Effect of temperature and pH on enzyme activity

    The effects of temperature on enzyme activity were determined by the incubation of the reaction mixtures at temperatures ranging from 30 to 80ºC in 50 mM Tris-HCl at pH 8.5. In order to determine the optimal pH for the enzyme activity, standard assay mixtures in the following buffers were utilized: 20 mM citrate buffer (pH 3.0-6.0), 20 mM sodium phosphate buffer (pH 6.0-7.6), 20 mM Tris-HCl (pH 7.5-9.0) and glycine-NaOH buffer (pH 10.0).


    Cloning and expression of the kdgK gene

    Sequence analysis showed that the presence of two open reading frames (ORFs), in which one ORF is maltose metabolism stimulation (mms) (Lee et al. 2000) and the other is a 2-keto-3-deoxygluconate kinase gene. Both showed opposite transcriptional directions. The KDGK ORF began with the initiation codon ATG at position 235 bp, located 10 bases downstream of the putative ribosomal binding site (AGGG), and the ending codon with the termination codon TAA at position 1160. The ATG codon was selected as the translation initiation site because its location was close to the possible ribosome binding site, 5’-AGGG-3’. The deduced KDGK amino acid sequence corresponds to a protein of 309 amino acids with a molecular weight of 33,993 Da. The putative RNA polymerase binding site evidenced the following homology to the classical consensus: 4 of 6 nt (TTGGCC) for the -35 region at position 121 and 4 of 6 nt (TAAAAC) for the -10 region at 126, with a spacing of 19 nt. At the 3’ end of the KDGK gene and surrounding the KDGK translational stop, there is a GC-rich imperfect inverted repeat (GCGGCGGCG-9nt-CGCCGCCC) followed by a run of A-T residues (TAATCAATAA). This sequence is typical for rho-independent transcription termination sites (Figure 2) (Von Hippel et al. 1984).

    Alignment of the KDGK operator with the consensus of the KdgR binding site

    Most of the KDGK genes regulated by the KdgR repressor harbor a typical KdgR-binding site (Reverchon et al. 1989). The consensus sequence of the KdgR binding site was detected at the 5’-untranslated end of KDGK. Sequence matching with the consensus 5’-AATAAAAC-xx-CGTTTCA-3’ of the KdgR binding site (Hugovieux-Cotte-Pattat et al. 1994) showed a 12/15 bp match between the consensus KdgR binding sequence and the S. marcescens binding site, 5’-ATTAAAAC-AC-TGTTTAA-3’ (Figure 3). This putative KdgR binding region partially overlaps the -10 element of the putative promoter region (Figure 2). This demonstrates that KdgR represses RNA polymerase for overlapping binding sites, thereby inhibiting transcriptional initiation.

    Amino acid sequence similarities between KDG kinase and other proteins

    Comparison of the KDG kinase with the NCBI databank revealed significant homology with an approximate 70% identity with the KDG kinase of Yersinia pseudotuberculosis, 69% identity with Erwinia chrysanthemi, and 68% identity with Salmonella enterica. These enzymes belong to the PfkB family among carbohydrate kinase families (Wu et al. 1991) (Figure 4).

    Purification of the recombinant enzyme

    The purification procedure is summarized in Table 1. E. coli cells harboring pKKDGK were expressed and evidenced increased enzyme activity after induction, but not the vector plasmid. The expressed enzyme was purified approximately 15 fold with an 11.8% recovery via GST affinity chromatography (Table 1). Subjecting the purified enzyme to SDS-PAGE generated a single protein band corresponding to a molecular mass of 34 kDa (Figure 5). Approximately 1.9 mg of the purified enzyme was acquired and the specific activity of the final preparation was estimated to be 12.6 U/mg (Table 1). The enzyme reaction was significant for both the substrates, 2-keto-gluconate (KG) and KDG. However, KDG proved to be approximately nine times (8.3) higher than KG (0.95) with regard to kinase specific activity.

    Effect of temperature and pH on KDG kinase activity

    When we evaluated the effects of temperature on KDGK activity, we detected maximum activity at 50ºC, and the activity declined suddenly at temperatures above 50ºC such that the activity at 80ºC was only approximately 20% that observed at 50ºC (Figure 6). When we determined the effects of pH on KDGK activity, we noted that the maximum activity was observed at pH 8.0, the activity declined slightly at pH values above 8.0, and very slight activity was noted at pH 5.0 and 6.0 (Figure 7).


    The KDG kinase is the key enzyme in the semi-phosphorylative ED pathway. This enzyme is a member of the ribokinase (PfkB) enzyme family, which is composed of prokaryotic sequences related to ribokinase, including enzymes such as fructokinases, the minor 6-phosphofructokinase of E. coli, 1-phosphofructokinase, and archaeal ADP-dependent glucokinases and phosphofructokinases (Ito et al. 2001). Thus far, the KDGK gene was cloned, expressed, and characterized in E. coli, Erwinia chrysanthemi, Sulfolobus tokodaii, and Sulfolobus solfataricus in the modified Entner-Doudoroff Pathway (Cynkin and Ashwell, 1960; Hugouvieux-Cotte-Pattat et al. 1994; Kim and Lee, 2006; Ohshima et al. 2007). The PfkB kinases are generally composed of 310 to 430 amino acid residues (Hugouvieux-Cotte-Pattat et al. 1994) and are distinguished by three domains (N terminal, middle, C terminal) (Henikoff and Henikoff, 1991). There are regions corresponding to the two signature patterns of carbohydrate kinases of the PfkB family. Consensus pattern 1 is [A,G]-G-[x]-[G,P]-x-N-x-[S,T,A]-6x-[G,S]-9x-G and consensus pattern 2 is [D,N,S]-[P,T,S]-x-[S,A,G]-[S,A,G]-G-D-3x-[A,G]-[A,G]-[L,I,V,M] -[L,I,V,M,S,T,A] among the consensus patterns of the PfkB family. Consensus pattern 2 was shown to be involved in ATP binding ability (Aulkemeyer et al. 1991). KDGK evidences significant homology, and approximately 70% identity with the KDG kinase of Yersinia pseudotuberculosis.

    The apparent molecular weight of the KDGK from Serratia marcescens KCTC 2172 was 34 kDa, similar to that from Sulfolobus tokodaii (Ohshima et al. 2007); a value of 35 kDa was detected from Sulfolobus solfataricus (Kim and Lee, 2006) and 160 kDa from Thermus thermophiles (Ohshima et al. 2004) In the hyperthermophile, Sulfolobus tokodaii, the maximum activity of the enzyme was observed at 80ºC, the activity declined rapidly at temperatures above 80ºC (Ohshima et al. 2007). The maximum enzyme activity was noted at between 70 and 80ºC and the optimal pH between 7.0 and 8.0 in S. sulfatoricus KDGK (Kim and Lee, 2006). In the present study, KDG kinase activity evidenced optimal activity at a temperature of 50ºC and a pH value of 8.0. In this study, we successfully expressed the Serratia marcescens KCTC 2172 gene and identified a product that evidenced KDGK activity.


    We thank Asahi Kasei Pharma in Tokyo and Professor Toshihisa Ohshima for the generous gift of ADP-dependent glucokinase.


    AULKEMEYER, P.; EBNER, R.; HEILENMANN, G.; JAHREIS, K.; SCHMID, K.; WRIEDEN, S. and LENGELER, J.W. Molecular analysis of two fructokinases involved in sucrose metabolism in enteric bacteria. Molecular Microbiology, December 1991, vol. 5, no. 12, p. 2913-2922. [CrossRef]        [ Links ]

    BRADFORD, Marion M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, May 1976, vol. 72, no. 1-2, p. 248-254. [CrossRef]        [ Links ]

    CONDEMINE, Guy; HUGOUVIEUX-COTTE-PATTAT, Nicole and ROBERT-BAUDOUY, Janine. Isolation of Erwinia chrysanthemi KduD mutants altered in pectin degradation. Journal of Bacteriology, March 1986, vol. 165, no. 3, p. 937-941.         [ Links ]

    CONDEMINE, Guy and ROBERT-BAUDOUY, Janine. Analysis of an Erwinia chrysanthemi gene cluster involved in pectin degradation. Molecular Microbiology, September 1991, vol. 5, no. 9, p. 2191-2202. [CrossRef]        [ Links ]

    CYNKIN, Morris A. and ASHWELL, Gilbert. Uronic acid metabolism in bacteria. IV. Purification and properties of 2-keto-3-deoxy-D-gluconokinase in Escherichia coli. Journal of Biological Chemistry, June 1960, vol. 235, no. 6, p. 1576-1579.        [ Links ]

    GAL, Sang Wan; CHOI, Ji Young; KIM, Cha Young; CHEONG, Young Hwa.; CHOI, Young Ju; LEE, Sang Yeol; BAHK, Jeong Dong and CHO, Moo Je. Cloning of the 52-kDa chitinase gene from Serratia marcescens KCTC2172 and its proteolytic cleavage into an active 35-kDa enzyme. FEMS Microbiology Letters, March 1998, vol. 160, no. 1, p. 151-158. [CrossRef]         [ Links ]

    GIRI, Anuradha V.; ANANDKUMAR, Nandini; MUTHUKUMARAN, Geetha and PENNATHUR, Gautam. A novel medium for the enhanced cell growth and production of prodigiosin from Serratia marcescens isolated from soil. BMC Microbiology, March 2004, vol. 4, p. 11. [CrossRef]        [ Links ]

    HENIKOFF, Steven and HENIKOFF, Jorga G. Automated assembly of protein blocks for database searching. Nucleic Acids Research, December 1991, vol. 19, no. 23, p. 6565-6572. [CrossRef]        [ Links ]

    HUGOUVIEUX-COTTE-PATTAT, Nicole and ROBERT-BAUDOUY, Janine. Isolation of kdgK-lac and kdgA-lac gene fusion in the phytopathogenic bacterium Erwinia chrysanthemi. Microbiology, May 1985, vol. 131, no. 5, p. 1205-1211.         [ Links ]

    HUGOUVIEUX-COTTE-PATTAT, Nicole and ROBERT-BAUDOUY, Janine. Hexauronate catabolism in Erwinia chrysanthemi. Journal of Bacteriology, March 1987, vol. 169, no. 3, p. 1223-1231.        [ Links ]

    HUGOUVIEUX-COTTE-PATTAT, Nicole and ROBERT-BAUDOUY, Janine. Isolation of Erwinia chrysanthemi mutantsaltered in pectinolytic enzyme production. Molecular Microbiology, November 1989, vol. 3, no. 11, p. 1587-1597. [CrossRef]        [ Links ]

    HUGOUVIEUX-COTTE-PATTAT, Nicole; NASSER, William and ROBERT-BAUDOUY, Janine. Molecular characterization of the Erwinia chrysanthemi kdgK gene involved in pectin degradation. Journal of Bacteriology, April 1994, vol. 176, no. 8, p. 2386-2392.        [ Links ]

    HUGOUVIEUX-COTTE-PATTAT, Nicole; CONDEMINE, Guy; NASSER, William and REVERCHON, Sylvie. Regulation of pectinolysis in Erwinia chrysanthemi. Annual Review of Microbiology, 1996, vol. 50, p. 213-257. [CrossRef]        [ Links ]

    ITO, Sohei; FUSHINOBU, Shinya; YOSHIOKA, Issei; KOGA, Shinji; MATSUZAWA, Hiroshi and WAKAGI, Takayoshi. Structural basis for the ADP-specificity of a novel glucokinase from a hyperthermophilic archaeon. Structure, March 2001, vol. 9, no. 3, p. 205-214. [CrossRef]        [ Links ]

    IWAMOTO, Ryoko and IMANAGA, Yujiro. Direct evidence of the Entner-Doudoroff pathway operating in the metabolism of D-glucosamine in bacteria. Journal of Biochemistry, January 1991, vol. 109, no. 1, p. 66-69.         [ Links ]

    KIM, Seonghun and LEE, Sun Bok. Characterization of Sulfolobus solfataricus 2-keto-3-deoxy-D-gluconate kinase in the modified Entner-Doudoroff pathway. Bioscience, Biotechnology and Biochemistry, 2006, vol. 70, no. 6, p. 1308-1316. [CrossRef]        [ Links ]

    LAEMMLI, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, August 1970, vol. 227, no. 5259, p. 680-685. [CrossRef]        [ Links ]

    LE, Thuc T.; GUET, Calin C. and CLUZEL, Philippe. Protein expression enhancement in efflux-deleted mutant bacteria. Protein Expression and Purification, July 2006, vol. 48, no. 1, p. 28-31. [CrossRef]        [ Links ]

    LEE, S.J.; SONG, O.R.; CHUNG, S.Y.; LEE, Y.C. and CHOI, Y.L. Characterization of polyphosphate kinase gene in Serratia marcescens. Journal of Life Science, 2000, vol.10, p. 397-402.         [ Links ]

    LEE, S.J.; YOO, J.S.; KIM, H.S.; LEE, S.C.; CHUNG, S.Y. and CHOI, Y.L. Analysis and cloning of the gene involved in activation of maltose metabolism in Serratia marcescens. Korean Journal of Applied Microbiology and Biotechnology, 2000, vol. 28, no. 1, p. 21-25.        [ Links ]

    LEE, Seung-Jin; LEE, Yong-Seok; LEE, Young-Choon and CHOI, Yong-Lark. Molecular characterization of polyphosphate (polyP) operon from Serratia marcescens. Journal of Basic Microbiology, April 2006, vol. 46, no. 2, p. 108-115. [CrossRef]        [ Links ]

    MANDRAND-BERTHELOT, Marie-Andree; RITZENTHALER, Paul and MATA-GILSINGER, Mireille. Construction and expression of hybrid plasmids containing the structural gene of the Escherichia coli K-12 3-deoxy-2-oxo-D-gluconate transport system. Journal of Bacteriology, November 1984, vol. 160, no. 2, p. 600-606.        [ Links ]

    NASSER, William; REVERCHON, Sylvie; CONDEMINE, Guy and ROBERT-BAUDOUY, Janine. Specific interactions of Erwinia chrysanthemi KdgR repressor with different operators of genes involved in pectinolysis. Journal of Molecular Biology, February 1994, vol. 236, no. 2, p. 427-440. [CrossRef]        [ Links ]

    OHSHIMA, N.; INAGAKI, E.; YASUIKE, K.; TAKIO, K.; TAHIROV, T.H. Structure of Thermus thermophilus2-keto-3-deoxygluconate kinase: evidence for recognition of an open chain substrate, Journal of Molecular Biology, 2004, vol. 340, p. 477-489.        [ Links ]

    OHSHIMA, Toshihisa; KAWAKAMI, Ryushi; KANAI, Yuichiro; GODA, Shuichiro and SAKURABA, Haruhiko. Gene expression and characterization of 2-keto-3-deoxygluconate kinase, a key enzyme in the modified Entner-Doudoroff pathway of the aerobic and acidophilic hyperthermophile Sulfolobus tokodaii. Protein Expression and Purification, July 2007, vol. 54, no. 1, p. 73-78. [CrossRef]         [ Links ]

    REVERCHON, Sylvie; HUANG, Yue; BOURSON, Claude and ROBERT-BAUDOUY, Janine. Nucleotide sequences of Erwinia chrysanthemi ogl and pelE genes negatively regulated by the kdgR product. Gene, December 1989, vol.85, no. 1, p. 125-134. [CrossRef]        [ Links ]

    VON HIPPEL, Peter H.; BEAR, David G.; MORGAN, William D. and MCSWIGGEN, James A. Protein-nucleic acid interaction in transcription: a molecular analysis. Annual Review of Biochemistry, July 1984, vol. 53, p. 389-446. [CrossRef]         [ Links ]

    WU, L.F.; REIZER, A.; REIZER, J.; CAI, B.; TOMICH, J.M. and SAIER, M.H. Nucleotide sequence of the Rhodobacter capsulatus fruK gene, which encodes fructose-1-phosphate kinase: evidence for a kinase superfamily including both phosphofructokinases of Escherichia coli. Journal of Bacteriology, May 1991, vol. 173, no. 10, p. 3117-3127.        [ Links ]

    YOO, Ju-Soon; KIM, Hae-Sun; CHUNG, Soo-Yeol and CHOI, Yong-Lark. Molecular characterization of crp, the cyclic AMP receptor protein gene of Serratia marcescens KTCC 2172. Journal of Microbiology and Biotechnology, 2000, vol. 10, no. 5, p. 670-676.         [ Links ]

    YOO, Ju Soon; KIM, Hae Sun; LEE, Young Choon; CHUNG, Soo Yeol and CHOI, Yong Lark. Molecular characterization of the genes encoding acetoacetyl-coenzyme A transferase from Serratia marcescens KCTC 2172. Journal of Microbiology and Biotechnology, 2001, vol. 11, no. 5, p. 870-875.         [ Links ]

    Note: Electronic Journal of Biotechnology is not responsible if on-line references cited on manuscripts are not available any more after the date of publication.

    Supported by UNESCO / MIRCEN network

    Creative Commons License Todo el contenido de esta revista, excepto dónde está identificado, está bajo una Licencia Creative Commons